Toxicity evaluation of metsulfuron-methyl, nicosulfuron, and methoxyfenozide as pesticides in Indonesia

Wahyu Daradjat Natawigena; Muhammad Ilfadry Rifasta; Agus Susanto; Gofarana Wilar; Cecep Suhandi

doi:10.1515/opag-2025-0443

Article Open Access

Toxicity evaluation of metsulfuron-methyl, nicosulfuron, and methoxyfenozide as pesticides in Indonesia

Wahyu Daradjat Natawigena , Muhammad Ilfadry Rifasta , Agus Susanto , Gofarana Wilar and Cecep Suhandi

Published/Copyright: May 6, 2025

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From the journal Open Agriculture Volume 10 Issue 1

Abstract

Pesticides are substances widely used to control or eliminate pests, including weeds, insects, and other harmful organisms. Agricultural pesticides, in particular, must meet specific safety and quality standards before being marketed. In this study, the acute and chronic toxicity of metsulfuron-methyl, nicosulfuron, and methoxyfenozide were predicted using quantitative structure–activity relationship (QSAR) analysis through the EPI Suite program. Furthermore, in vivo testing was conducted to evaluate skin irritation, eye irritation, and skin sensitization based on modified OECD 404, OECD 405, and OECD 406 guidelines. The QSAR analysis indicated that all three pesticides have low acute and chronic toxicity profiles. In the skin irritation and skin sensitization tests, metsulfuron-methyl (100 mg), nicosulfuron (125 mg), and methoxyfenozide (150 mg) showed a primary irritation score of 0 and a sensitization score of 0, indicating no irritant or sensitization effects on rabbit and guinea pig skin. In the eye irritation test, metsulfuron-methyl caused mild conjunctival redness and eyelid swelling, with an average irritation score of 1.25, classifying it as a mild irritant (Category 2B). In contrast, nicosulfuron and methoxyfenozide did not cause any observable eye irritation, with an irritation score of 0. These findings suggest that while metsulfuron-methyl may cause mild eye irritation, all three pesticides are non-irritating to the skin and do not induce skin sensitization. This research provides essential toxicological data for regulatory safety evaluations and underscores the importance of integrating QSAR analysis with in vivo methods to assess pesticide safety comprehensively.

Keywords: irritation test; sensitization test; metsulfuron-methyl; nicosulfuron; methoxyfenozide; toxicity

1 Introduction

Pesticides, including herbicides and insecticides, play a crucial role in modern agricultural practices by protecting crops from pests and weeds, thereby increasing yields and ensuring food security [1]. However, their extensive use raises significant concerns about environmental pollution and human health risks due to potential toxicity [2]. According to the World Health Organization (WHO), approximately 18.2 out of every 100,000 farmers worldwide experience pesticide poisoning, with more than 168,000 deaths occurring annually as a result of pesticide exposure [3]. These cases are predominantly reported in developing countries. Research conducted by Boedeker et al. estimates that 385 million cases of unintentional acute pesticide poisoning (UAPP) occur globally each year, resulting in approximately 11,000 deaths. Given that the global farming population is around 860 million, these figures indicate that approximately 44% of the farmers experience pesticide poisoning annually [4]. The highest number of UAPP cases is recorded in regions such as Southern Asia, Southeast Asia, and East Africa, where non-fatal poisonings are most prevalent.

Although botanical insecticides are considered to have fewer adverse effects on the environment, they decompose quickly, resulting in a shorter shelf life and slower efficacy compared to synthetic insecticides. Consequently, synthetic insecticides are more widely used due to their rapid action, ease of availability, and immediate effectiveness, despite posing a greater risk to environmental and human health [5]. Among the various herbicides and insecticides, metsulfuron-methyl, nicosulfuron, and methoxyfenozide are extensively used because of their effectiveness in controlling specific agricultural pests [5].

Metsulfuron-methyl is an active ingredient in a systemic herbicide that effectively suppresses the dry weight accumulation of broadleaf weeds and other weeds for up to 12 weeks after application. It is commonly used to control weeds in rice fields [6,7]. Nicosulfuron is a selective herbicide that targets a wide range of weeds, including grass, broadleaf, and sedge weeds [8,9]. Methoxyfenozide is an insect growth regulator (IGR) that functions as an ecdysone agonist, interfering with insect development and growth. It is particularly effective against Lepidoptera pests. Methoxyfenozide belongs to the diacylhydrazine class of insecticides, which bind with high affinity to the ecdysone receptor complex and act as strong agonists or mimics of the insect molting hormone, 20-hydroxyecdysone (20E) [10,11].

Despite their agricultural benefits, there is a critical need to assess the toxicity of these compounds to ensure human and environmental safety while meeting regulatory compliance standards. Previous research on metsulfuron-methyl and nicosulfuron has primarily focused on their herbicidal efficacy and environmental persistence [4]. Meanwhile, methoxyfenozide has been studied for its effects on insect development, but limited information is available regarding its potential for skin and eye irritation or sensitization risks [12,13]. Furthermore, there is a notable research gap in integrating quantitative structure–activity relationship (QSAR)-based predictions to complement traditional toxicity evaluations of these substances. By incorporating QSAR analysis, our study addresses this gap, providing a more comprehensive assessment of the acute and chronic effects of these pesticides.

The primary objective of this study was to evaluate the skin irritation, eye irritation, and skin sensitization potential of metsulfuron-methyl, nicosulfuron, and methoxyfenozide using in vivo methods in compliance with established OECD guidelines. To enhance the comprehensiveness of our findings, we also performed QSAR analysis using the EPI Suite program to predict their acute and chronic toxicity profiles. This integrated experimental and computational approach aims to provide a robust assessment of these pesticides’ safety, offering valuable insights for regulatory agencies and guiding future research.

2 Materials and methods

2.1 Chemicals and reagents

The pesticides used in this study and their purity are as follows:

MEVOR 20 WP (active ingredient: metsulfuron-methyl, 20%; provided by PT. Sari Kresna Kimia, Indonesia).
FURON 250 SC (active ingredient: nicosulfuron, 250 g/L; provided by PT. Sari Kresna Kimia, Indonesia).
METRICOR 300 SC (active ingredient: methoxyfenozide, 300 g/L; provided by PT. Sari Kresna Kimia, Indonesia).

All reagents used were of analytical grade and prepared according to OECD guidelines.

2.2 Computational analysis of environmental fate and toxicity estimation

The environmental fate and toxicity of three pesticides – metsulfuron-methyl, nicosulfuron, and methoxyfenozide – were evaluated using the United States Environmental Protection Agency’s (USEPA) EPI Suite™ version 4.11 (EPI = estimation program interface) [14]. This software estimates key physicochemical properties and environmental behaviors, including partition coefficients, biodegradability, aquatic toxicity, atmospheric degradation, and environmental distribution via a fugacity model [15,16,17,18].

2.2.1 Data input

For each pesticide, the analysis was performed by entering either the Chemical Abstract Service (CAS) number or the chemical structure. The CAS numbers used were as follows: metsulfuron-methyl (74223-64-6), nicosulfuron (111991-09-4), and methoxyfenozide (161050-58-4). The program automatically retrieved the molecular structures and relevant data from its internal database.

2.2.2 Parameter settings

To simulate environmental conditions specific to aquatic ecosystems, the following key environmental parameters were defined:

Water depth: 2 m for rivers and 20 m for lakes, based on typical hydrological conditions in Indonesia [19,20].
Wind velocity: 2 m/s for rivers and 1 m/s for lakes, representing average atmospheric conditions [21].
Current velocity: 0.2 m/s for rivers and 0.02 m/s for lakes to simulate water movement and mixing dynamics [19,22].

These parameters were selected based on environmental studies of Indonesian aquatic ecosystems to enhance the accuracy of the simulation.

2.2.3 Modules applied

The following EPI Suite modules were employed:

KOWWIN: estimates the octanol–water partition coefficient (log Kow).
BIOWIN: predicts the aerobic biodegradability using multiple models.
ECOSAR: estimates the acute and chronic aquatic toxicity to fish, daphnids, and algae.
HYDROWIN: calculates the hydrolysis rate under neutral pH conditions.
AEROWIN: predicts atmospheric oxidation and Henry’s law constant (HLC) for assessing volatilization.
BCFBAF: estimates the bioconcentration factor (BCF) to evaluate the bioaccumulation potential.
EQC (fugacity model): estimates the chemical distribution across environmental compartments (air, water, soil, and sediment) using a Level III fugacity model.

2.2.4 Output and data analyses

The results were exported to plain text (.txt) files for each pesticide. Key parameters, including log Kow, biodegradation potential, aquatic toxicity (LC₅₀ and EC₅₀ values), bioaccumulation factor, and atmospheric half-life, were extracted and analyzed. Additionally, the EQC model provided quantitative estimates of the percentage distribution in air, water, soil, and sediment, reflecting each pesticide’s environmental partitioning behavior [14]. The data were interpreted according to US EPA toxicity classification guidelines and compared across the three pesticides to assess their relative environmental impact and persistence.

2.3 In vivo toxicological studies

2.3.1 Animal model and ethical approval

The study used New Zealand albino rabbits for skin and eye irritation tests and Dunkin Hartley albino guinea pigs for the skin sensitization test. All animals were sourced from certified breeders, housed under controlled environmental conditions (22 ± 2°C, 55 ± 10% relative humidity, and 12-h light/dark cycle), and provided with food and water ad libitum.

Ethical approval: The study followed ethical guidelines for animal welfare and was approved by the Research Ethics Committee of Universitas Padjadjaran, Bandung (protocol code 582/UN6.KEP/EC/2023, approved on May 6, 2023).

2.3.2 Skin irritation tests

Skin irritation tests were conducted following the OECD guideline 404 [3]. New Zealand albino rabbits were used for the test, and their fur was shaved the day before the procedure. The test began with an initial assessment on one rabbit, where 0.5 g of solid samples or 0.5 mL of liquid samples were applied. The doses used for each pesticide were as follows: metsulfuron-methyl (100 mg), nicosulfuron (125 mg), and methoxyfenozide (150 mg). Three patches containing the test sample and one control patch (0.9% NaCl) were used. The patches were sequentially removed after 3 min, 1 h, and 4 h. The irritation response was evaluated at 24, 48, and 72 h after exposure, with further observations for reversibility conducted on days 7 and 14.

To confirm the initial findings, a confirmation test was performed using three additional rabbits. In this phase, two patches (one containing the sample and one containing control – 0.9% NaCl) were applied, and the sample patch was removed after 4 h. Similar to the initial test, the irritation response was assessed at 24, 48, and 72 h, with reversibility observed on days 7 and 14. Irritation responses were evaluated according to the Draize skin irritation test rating scale (Table 1) and classified based on the skin irritation classification criteria (Table 2) [3,23].

Primary irritation score = ( A − B ) / C ,

where A is the total score of edema and erythema during observation; B is the number of observation periods; C is the number of rabbits in the group.

Table 1

Draize skin irritation test scoring [3,24]

Skin reaction	Score
Erythema and eschar formation
No erythema	0
Very slight erythema	1
Well-defined erythema	2
Moderate to severe erythema	3
Edema formation
No edema	0
Very slight edema	1
Slight edema	2
Moderate edema	3

Table 2

Classification of skin irritation [3,23]

Classification	Primary irritation score
No erythema	0
Very slight erythema	<2
Well-defined erythema	2–5
Moderate to severe erythema	>5

2.3.3 Eye irritation tests

Eye irritation tests were conducted following the OECD guideline 405. New Zealand albino rabbits were used for the test. An initial test was performed using one rabbit per sample. The sample was applied to the left eye at a dose of 0.1 g for solid samples and 0.1 mL for liquid samples. The doses used for each pesticide were as follows: metsulfuron-methyl (20 mg), nicosulfuron (25 mg), and methoxyfenozide (30 mg). After 1 h of exposure, the treated eye was carefully rinsed with sterile saline solution. Observations were recorded at 1 h, 24 h, 48 h, 72 h, 7 days, 14 days, and 21 days after exposure. If no severe eye irritation was observed during the initial test, a confirmation test was performed using three additional rabbits to validate the results. All observations were evaluated using the Draize eye irritation test scoring system (Table 3) [25] and classified according to the eye irritation category (Table 4) [26].

Table 3

Draize eye irritation test scoring [25,27]

Endpoint	Description	Score range
Cornea	Degree of opacity and ulcerations	0–4
Iris	Swelling and hyperemia	0–2
Conjunctivae	Redness and vessel discernibility	0–3
Chemosis	Swelling and lids closed/open	0–4

Table 4

Category of eye irritation [26]

Classification	GHS classification criteria
Category 1 (causes serious eye damage)	Classification as corrosive to the skin; Human experience or data showing damage to the eye which is not fully reversible within 21 days; Structure/activity or structure/property relationship to a substance or mixture already classified as corrosive; pH extremes of <2 and >11.5 including the buffering capacity; Positive results in a valid and accepted in vitro test to assess serious damage to the eyes; or Animal experience or test data that the substance or mixture produces either (1) in at least one animal, effects on the cornea, iris or conjunctiva that are not expected to reverse or have not reversed; or (2) in at least 2 of 3 tested animals a positive response of corneal opacity ≥ 3 and/or iritis > 1.5, calculated as the mean scores, following grading at 24, 48, and 72 h
Category 2A (irritant)	Classification as severe skin irritant; Human experience or data showing changes in the eye which are fully reversible within 21 days; Structure/activity or structure property relationship to a substance or mixture already classified as an eye irritant; Positive results in a valid and accepted in vitro eye irritation test; or Animal experience or test data that indicate that the substance/mixture produces a positive response in at least 2 of 3 tested animals of corneal opacity ≥ 1, iritis ≥ 1, or conjunctival edema (chemosis) ≥ 2, calculated as the mean scores, following grading at 24, 48, and 72 h and the effects are reversible within 21 days.
Category 2B (mild irritant)	Human experience or data showing production of mild eye irritation; If a substance meets classification criteria for 2A and such effects can be reversible within <strong> 7 days, </strong> the substance can be classified as 2B.

2.3.4 Skin sensitization tests

The skin sensitization test was conducted following the OECD guideline 406 using Dunkin Hartley albino guinea pigs. The doses used for each pesticide were 100 mg of metsulfuron-methyl, 125 mg of nicosulfuron, and 150 mg of methoxyfenozide. To determine the appropriate concentration, an initial test was performed on one guinea pig for each pesticide, exposing it to concentrations of 25, 50, 75, and 100% of the respective dose for 6 h. In the main test, three guinea pigs were used and exposed to the highest concentration that did not cause erythema during the initial test. Exposure was carried out on days 0, 6, and 13, followed by a challenge test on day 27, with observations recorded at 24 and 48 h post-exposure. The skin reactions were evaluated using the Magnusson–Kligman scale (Table 5) [28].

Table 5

Magnusson–Kligman scaling for determining the sensitization reaction [28,29]

Reaction	Grading scale
No visible change	0
Discrete or patchy erythema	1
Moderate and confluent erythema	2
Intense erythema and swelling	3

3 Results

3.1 Predicted environmental partitioning and toxicity outcomes

The in silico analysis using EPI Suite provided comprehensive predictions regarding the environmental fate and toxicity of metsulfuron-methyl, nicosulfuron, and methoxyfenozide across several parameters (Table 6).

Table 6

Predicted acute and chronic toxicity of metsulfuron-methyl, nicosulfuron, and methoxyfenozide using QSAR analysis (EPI Suite program)

EPI Suite program	Parameter	Results
EPI Suite program	Parameter	Metsulfuron-methyl	Nicosulfuron	Methoxyfenozide
ECOSAR	LC₅₀ (fish) (mg/L)	311.606	2.26 × 10⁵	14.303
	LC₅₀ (daphnid) (mg/L)	176.974	95,876.414	9.307
	EC₅₀ (green algae) (mg/L)	131.960	21,484.348	12.179
BIOWIN	BIOWIN 3	2.1178	1.9076	1.8901
BIOWIN	BIOWIN 5	0.5896	0.2512	−0.2077
KOWWIN	Log Kow	2.0026	−1.15	3.476
BCFBAF	BCF (L/kg wet-wt.)	3.996	3.162	128.3
HYDROWIN	Half-life in pH = 4–5 (days)	Less than 1–20	Less than 1–20	300+
	Half-life in pH = 7 (days)	100–300+	100–300+	300+
	Half-life in pH = 9 (days)	100–300+	100–300+	300+
AEROWIN	HLC (atm-m³/mol)	7.52 × 10⁻¹⁴	1.39 × 10⁻¹⁸	3.84 × 10⁻¹²
EQC (fugacity model)	Distribution	Soil	Soil	Soil

In the ECOSAR module, the estimated acute toxicity (LC₅₀) for fish varied significantly across the three pesticides [30]. Metsulfuron-methyl showed a moderate toxicity value of 311.606 mg/L, while nicosulfuron displayed the lowest toxicity with a value of 2.26 × 10⁵ mg/L. In contrast, methoxyfenozide exhibited the highest toxicity to fish at 14.303 mg/L. A similar pattern was observed for daphnids, where metsulfuron-methyl had an LC₅₀ of 176.974 mg/L, nicosulfuron reached 95,876.414 mg/L, and methoxyfenozide showed the highest toxicity with 9.307 mg/L. Regarding green algae, metsulfuron-methyl demonstrated an EC₅₀ of 131.960 mg/L, nicosulfuron showed 21,484.348 mg/L, while methoxyfenozide exhibited the most potent toxicity at 12.179 mg/L.

The BIOWIN module estimated the biodegradability of the compounds [31]. For metsulfuron-methyl, the BIOWIN 3 and BIOWIN 5 scores were 2.1178 and 0.5896, respectively, indicating moderate biodegradability. Nicosulfuron presented slightly lower values at 1.9076 (BIOWIN 3) and 0.2512 (BIOWIN 5), suggesting a slower degradation rate. Methoxyfenozide showed the lowest biodegradability potential with BIOWIN 3 at 1.8901 and BIOWIN 5 at −0.2077, indicating higher environmental persistence.

According to KOWWIN results, the log Kow values varied, reflecting differences in hydrophobicity [32]. Metsulfuron-methyl had a log Kow of 2.0026, suggesting moderate lipophilicity, while nicosulfuron displayed a negative value (−1.15), indicating high hydrophilicity. Methoxyfenozide had the highest log Kow value of 3.476, implying greater potential for bioaccumulation.

The BCFBAF module estimated the BCF in aquatic organisms [33]. Metsulfuron-methyl and nicosulfuron demonstrated relatively low bioaccumulation potentials with BCF values of 3.996 L/kg wet-wt. and 3.162 L/kg wet-wt., respectively. Methoxyfenozide, however, displayed a significantly higher BCF of 128.3 L/kg wet-wt., suggesting a greater likelihood of bioaccumulation.

The HYDROWIN module predicted the hydrolysis rates under different pH conditions [34]. At pH 4–5, both metsulfuron-methyl and nicosulfuron exhibited a half-life of less than 1–20 days, suggesting rapid degradation. In contrast, methoxyfenozide showed a more extended half-life of 300+ days, indicating higher stability. At pH 7 and pH 9, all three pesticides exhibited extended degradation times, with half-lives ranging from 100 to 300+ days.

The AEROWIN module calculated the HLC, which indicates volatility [35]. Metsulfuron-methyl had an HLC value of 7.52 × 10⁻¹⁴ atm-m³/mol, while nicosulfuron displayed the lowest volatility at 1.39 × 10⁻¹⁸ atm-m³/mol. Methoxyfenozide exhibited the highest volatility among the three compounds with 3.84 × 10⁻¹² atm-m³/mol.

Finally, the EQC (fugacity model) results indicated that all three pesticides are predominantly distributed in the soil compartment under environmental conditions, suggesting limited mobility to other environmental matrices like air or water.

3.2 Skin irritation tests

In the initial skin irritation test for metsulfuron-methyl, nicosulfuron, and methoxyfenozide, no signs of irritation, such as erythema or edema, were observed at 3 min, 1, and 4 h after administration. Observations continued at 24, 48, and 72 h, during which time no irritation reactions were detected for any of the three samples, as shown in Table 7. Based on these initial findings, skin irritation was further assessed by calculating the primary irritation score.

Table 7

Skin irritation initial test results

Sample	Reaction	Score
Sample	Reaction	0 h	4 h	24 h	48 h	72 h
Metsulfuron-methyl	Erythema and eschar formation	0	0	0	0	0
Metsulfuron-methyl	Edema formation	0	0	0	0	0
Primary irritation score		0
Nicosulfuron	Erythema and eschar formation	0	0	0	0	0
Nicosulfuron	Edema formation	0	0	0	0	0
Primary irritation score		0
Methoxyfenozide	Erythema and eschar formation	0	0	0	0	0
Methoxyfenozide	Edema formation	0	0	0	0	0
Primary irritation score		0

From the initial test data and the primary irritation score in Table 7, all three samples – metsulfuron-methyl, nicosulfuron, and methoxyfenozide – received a score of 0, indicating no irritation. An example of these results is shown in Figure 1. To confirm the initial findings, a confirmation test was performed. The confirmation test was conducted to verify the absence of irritation observed in the initial test. This phase involved three New Zealand albino rabbits, with exposure to the samples for 4 h. Observations were conducted at 24, 48, and 72 h to assess the irritation index and continued on days 7 and 14 to evaluate reversibility.

Figure 1

Example of the results of an initial test of skin irritation test that has been exposed for 4 h (3’: 3 min exposure; 1°: 1 h exposure; 4°: 4 h exposure; C: control area).

As shown in Table 8, the primary irritation score from the confirmation test for all three samples remained 0, indicating no skin irritation. Based on these results, metsulfuron-methyl, nicosulfuron, and methoxyfenozide were classified as non-irritants according to the Draize skin irritation test criteria.

Table 8

Skin irritation confirmation test results

Sample		Reaction	Score
Sample		Reaction	0 h	4 h	24 h	48 h	72 h
Metsulfuron-methyl	x̄ 3 Rabbit	Erythema and eschar formation	0	0	0	0	0
Metsulfuron-methyl	x̄ 3 Rabbit	Edema formation	0	0	0	0	0
	Primary irritation score		0
Nicosulfuron	x̄ 3 Rabbit	Erythema and eschar formation	0	0	0	0	0
Nicosulfuron	x̄ 3 Rabbit	Edema formation	0	0	0	0	0
	Primary irritation score		0
Methoxyfenozide	x̄ 3 Rabbit	Erythema and eschar formation	0	0	0	0	0
Methoxyfenozide	x̄ 3 Rabbit	Edema formation	0	0	0	0	0
	Primary irritation score		0

No erythema or edema was observed 4 h after the test preparations were applied. Further observations at 24 h after patch removal revealed no signs of irritation. Monitoring continued until day 7, and no irritation symptoms were detected. By day 14, no erythema or edema was observed, and fur regrowth was noted at the test sites. Typically, complete fur regrowth in rabbits occurs within 30 days of testing. Throughout the observation period, no irritation responses were noted, and the skin fully recovered without signs of lasting damage. Based on these findings, it was concluded that metsulfuron-methyl, nicosulfuron, and methoxyfenozide do not cause skin irritation and are classified as non-irritants. An example of the results is shown in Figure 2.

Figure 2

Example of the results of a confirmation test of skin irritation test that has been exposed for 4 h (4°: 4 h exposure; C: control area).

3.3 Eye irritation test

In the initial eye irritation test, one New Zealand albino rabbit was used for each pesticide sample. Observations were conducted at 24, 48, and 72 h after exposure. If irritation was observed, further evaluations were carried out on days 7, 14, and 21 to assess the reversibility, healing time, or delayed onset of irritation. The severity of eye irritation was evaluated using the Draize eye irritation test scoring system, as outlined in Table 9.

Table 9

Eye irritation initial test results

Sample	Iritation reaction	Score
Sample	Iritation reaction	1 h	24 h	48 h	72 h
Metsulfuron-methyl	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	1	1	0	0
	Chemosis	1	1	1	0
Nicosulfuron	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	0	0	0	0
	Chemosis	0	0	0	0
Methoxyfenozide	Cornea	0	0	0	0
	Iris	0	0	0	0
	Conjunctivae	0	0	0	0
	Chemosis	0	0	0	0

According to the initial eye irritation test results in Table 9, the metsulfuron-methyl sample caused chemosis, characterized by mild redness of the conjunctiva and slight swelling of the eyelids, with an average score of 1.25. However, by 72 h, and at subsequent observations on days 7, 14, and 21, complete healing was observed. In contrast, the nicosulfuron and methoxyfenozide samples did not produce any observable irritation. The treated eyes were identical to the control eyes, with no swelling of the iris or eyelids. Even during prolonged observation on days 7, 14, and 21, no irritation was detected, with a consistent score of 0. Based on these findings, metsulfuron-methyl was classified as a mild irritant, while nicosulfuron and methoxyfenozide were classified as non-irritants. An example of the initial test results is shown in Figure 3.

Figure 3

Comparison of the eye exposed to metsulfuron-methyl with the control eye in the first hour of the initial test: (a) eye exposed by metsulfuron-methyl; (b) control eye.

To confirm these findings, a confirmation eye irritation test was conducted using three additional New Zealand albino rabbits for each pesticide. The procedures mirrored those of the initial test, with observations performed at 24, 48, and 72 h, followed by evaluations on days 7, 14, and 21 to monitor the severity and reversibility of eye irritation. As shown in Table 10, rabbits exposed to the metsulfuron-methyl sample exhibited slight eyelid swelling compared to the control eye. However, the cornea remained clear, and the iris and conjunctiva appeared normal, resulting in an average irritation score of 0.325. By 48 h, and throughout subsequent observations until day 21, full recovery was observed. Based on these findings, metsulfuron-methyl is classified as a Category 2B (mild irritant) according to the OECD guidelines. The results of the confirmation test are shown in Figure 4. In contrast, the nicosulfuron and methoxyfenozide samples did not induce any observable irritation. At all observation points – 1, 24, 48, and 72 h – the treated eyes remained identical to the control eyes, with no swelling or other signs of irritation. Continued monitoring on days 7, 14, and 21 confirmed the absence of delayed irritation. Therefore, nicosulfuron and methoxyfenozide were classified as non-irritants.

Table 10

Eye irritation confirmation test results

Sample	Rabbit	Iritation reaction	Score
Sample	Rabbit	Iritation reaction	1 h	24 h	48 h	72 h
Metsulfuron-methyl	x̄ 3 Rabbit	Cornea	0	0	0	0
		Iris	0	0	0	0
		Conjunctivae	0	0	0	0
		Chemosis	1	0.3	0	0
Nicosulfuron	x̄ 3 Rabbit	Cornea	0	0	0	0
		Iris	0	0	0	0
		Conjunctivae	0	0	0	0
		Chemosis	0	0	0	0
Methoxyfenozide	x̄ 3 Rabbit	Cornea	0	0	0	0
		Iris	0	0	0	0
		Conjunctivae	0	0	0	0
		Chemosis	0	0	0	0

Figure 4

Comparison of the eye exposed to metsulfuron-methyl with the control eye in the first hour of confirmation test: (a) eye exposed by metsulfuron-methyl; (b) control eye.

3.4 Skin sensitization test

The concentration determination for the sensitization test was performed using one Dunkin Hartley albino guinea pig for each pesticide sample. The concentration used for induction exposure was the highest concentration that caused mild irritation, while the concentration for further exposure was the highest concentration that did not cause irritation. These concentrations were determined through preliminary tests using three test animals. The initial concentrations tested were 12.5, 25, 50, 75, and 100%, with each exposure lasting for 6 h, followed by observations at 24 and 48 h after the gauze was removed. The results were evaluated according to the Magnusson and Kligman Scale presented in Table 11.

Table 11

Concentration determination results of the sensitization test based on the Magnusson–Kligman scale

Sample	Concentration	Irritation reaction
Metsulfuron-methyl	Control	0
	12.5%	0
	25%	0
	50%	0
	75%	0
	100%	0
Nicosulfuron	Control	0
	12.5%	0
	25%	0
	50%	0
	75%	0
	100%	0
Methoxyfenozide	Control	0
	12.5%	0
	25%	0
	50%	0
	75%	0
	100%	0

Based on the results in Table 11, guinea pigs exposed to metsulfuron-methyl at 100% concentration for 6 h did not show any signs of irritation when observed at 24 and 48 h after the gauze was removed, with a recorded irritation score of 0 across all concentrations. Similarly, nicosulfuron and methoxyfenozide samples, when applied at the highest concentration of 100%, also did not cause any irritation during the same observation period, with a score of 0 for both. As no irritation was observed in any of the samples, a 100% concentration was selected for use in the confirmation test. An example of the preliminary test results is shown in Figure 5.

Figure 5

Example of the results of concentration determination of the skin irritation test with 5 concentration variations and 1 control area that has been exposed for 6 h.

In the main sensitization test, three Dunkin Hartley albino guinea pigs were used for each pesticide sample. Each guinea pig was topically induced with a 100% concentration of metsulfuron-methyl, nicosulfuron, or methoxyfenozide on days 0, 6, and 13. This process was intended to trigger an immunological response that could lead to skin sensitization. On day 27, a challenge test was conducted to determine whether any observed reactions were due to sensitization rather than irritation.

The results presented in Table 12 indicated that none of the pesticide samples caused sensitization reactions. Metsulfuron-methyl, when applied at 100% concentration, did not produce any signs of skin sensitization at 24 and 48 h after the challenge test. The test area was compared with the control area exposed to 0.9% NaCl, and no differences were observed, with a recorded score of 0 across all samples. Similarly, nicosulfuron and methoxyfenozide did not produce any sensitization reactions, as the test areas remained indistinguishable from the control areas throughout the observation period.

Table 12

Main test observation after the challenge test

Sample	Guinea pig	Score
Sample	Guinea pig	6 h	24 h	48 h
Metsulfuron-methyl	Guinea pig A	0	0	0
	Guinea pig B	0	0	0
	Guinea pig C	0	0	0
Nicosulfuron	Guinea pig A	0	0	0
	Guinea pig B	0	0	0
	Guinea pig C	0	0	0
Methoxyfenozide	Guinea pig A	0	0	0
	Guinea pig B	0	0	0
	Guinea pig C	0	0	0

Further analysis confirmed that metsulfuron-methyl, nicosulfuron, and methoxyfenozide did not induce any skin sensitization responses in Dunkin Hartley albino guinea pigs. Therefore, it can be concluded that these pesticides do not have potential to cause skin sensitization. According to the Magnusson and Kligman scale, metsulfuron-methyl, nicosulfuron, and methoxyfenozide are classified as non-sensitizers. An example of the sensitization test results is shown in Figure 6.

Figure 6

Example of the results of a challenge test of skin sensitization test that has been exposed for 6 h (S: sample area; C: control area).

4 Discussion

Pesticides must meet quality standards, demonstrate effectiveness, and be safe for humans and the environment. To obtain regulatory approval and product certification, comprehensive toxicological evaluations are required. These studies provide crucial data for risk assessment and clinical management, supporting policy decisions by regulatory agencies for pesticide approval and categorization. Moreover, limiting the availability of highly toxic pesticides through licensing and registration could significantly reduce poisoning incidents without compromising agricultural productivity [12,13].

One major concern regarding pesticide exposure is the potential to cause skin and eye irritation or skin sensitization. Improper use of pesticides, such as the absence of personal protective equipment (PPE) during spraying, increases the risk of exposure. This can lead to symptoms of skin irritation, including itching, redness, dryness, swelling, and blisters [36,37]. The inflammatory response caused by chemical exposure is characterized by erythema, edema, and warmth due to vasodilation and plasma leakage [38,39]. Skin sensitization, on the other hand, is an immunological reaction triggered by certain chemicals, manifesting as pruritis, erythema, or edema [40,41]. Pesticide exposure can also affect ocular tissues, causing inflammation, redness, and, in severe cases, vision impairment or chemosis [42,43,44].

Toxicological evaluation typically involves both in vivo and in vitro methods. While in vitro alternatives, such as OECD-validated assays, are increasingly used to reduce animal testing, in vivo methods remain the gold standard for assessing complex biological interactions and systemic toxicity [45]. In this study, the QSAR analysis using the EPI Suite program predicted that metsulfuron-methyl, nicosulfuron, and methoxyfenozide exhibit low acute and chronic toxicity, suggesting minimal potential for causing skin irritation, eye irritation, and skin sensitization. These computational predictions were validated through in vivo tests conducted according to modified OECD 404, OECD 405, and OECD 406 guidelines [46].

In the skin irritation test, herbicide samples containing metsulfuron-methyl (200 g/L) and nicosulfuron (250 g/L) and the insecticide containing methoxyfenozide (300 g/L) exhibited a primary irritation score of 0, indicating that these substances do not cause skin irritation in rabbits. This suggests that, when applied topically, these pesticides are unlikely to induce immediate or delayed skin reactions [11,47]. These findings align with the existing literature, which indicates low dermal toxicity for sulfonylurea herbicides and insecticides like methoxyfenozide [48,49,50]. This is particularly important for occupational safety, as skin contact is a common route of pesticide exposure among agricultural workers [51,52].

In the eye irritation test, metsulfuron-methyl caused mild conjunctival redness and eyelid swelling, with an average irritation score of 0.325, classifying it as a mild irritant (Category 2B). However, the symptoms were reversible by the 21st day of observation, indicating no long-term ocular damage. In contrast, nicosulfuron and methoxyfenozide did not cause any observable eye irritation throughout the observation period, with an irritation score of 0. This suggests that while metsulfuron-methyl poses a minor risk of temporary eye irritation, the other two pesticides are safer for ocular exposure under normal usage conditions.

The skin sensitization test revealed no erythema or edema reactions in guinea pigs exposed to the highest concentration (100%) of each pesticide. This indicates that metsulfuron-methyl, nicosulfuron, and methoxyfenozide do not induce skin sensitization, supporting their classification as non-sensitizing substances. These findings are consistent with previous studies, reporting low immunogenic potential for sulfonylurea herbicides and methoxyfenozide.

The consistency between QSAR predictions and in vivo findings in this study reinforces the reliability of computational models for preliminary toxicological assessments. This approach not only reduces the need for extensive animal testing but also provides rapid insights into potential health risks [53]. Furthermore, the data generated from this study contribute valuable toxicological information for regulatory safety evaluations and the assessment of occupational and environmental exposure risks.

Our findings are consistent with the existing literature on the safety of these pesticides. A study on methoxyfenozide’s environmental behavior and application in controlling litchi and longan pests reported low toxicity to non-target organisms, which aligns with our observations of its minimal skin and eye irritation potential [54]. For metsulfuron-methyl and nicosulfuron, while specific studies on their dermal and ocular effects are limited, previous research indicates that sulfonylurea herbicides exhibit low mammalian toxicity, supporting our findings of negligible skin irritation and sensitization [55,56]. These results are particularly relevant for regulatory safety evaluations, providing additional evidence to support the safe use of these pesticides when handled appropriately.

However, this study has some limitations. Notably, no positive control was used in the irritation and sensitization tests, as this study was intended as a preliminary assessment. The absence of a positive control, such as hexyl cinnamic aldehyde, limits the ability to benchmark the responses against known irritants or sensitizers. Future studies will incorporate positive controls to further validate these findings and provide a more comprehensive risk assessment. Additionally, while acute toxicity and immediate effects were evaluated, further research is needed to explore chronic toxicity, environmental impact, and the transformation products of these pesticides. Expanding the study to real-environment samples would provide more robust insights into human and ecological safety, addressing the complexities of pesticide exposure in agricultural and environmental contexts.

Moreover, this study serves as a foundation for future work that will incorporate modern in vitro methodologies for further verification and a more comprehensive assessment of the safety profiles of these pesticides. Future research should adopt OECD-accepted in vitro methods where applicable, aligning with the 3Rs principle (Replacement, Reduction, and Refinement) to minimize animal use while ensuring robust toxicological evaluations. This integrated approach will enhance both the accuracy and ethical standards of future toxicity studies.

5 Conclusions

This research evaluated the toxicity of herbicides containing metsulfuron-methyl (200 g/L), nicosulfuron (250 g/L), and the insecticide methoxyfenozide (300 g/L) through QSAR analysis and in vivo testing. QSAR analysis indicated that all three pesticides exhibit low acute and chronic toxicity, suggesting minimal risk for skin and eye irritation and skin sensitization. Consistent with these predictions, the skin irritation test showed a primary irritation score of 0 for all three samples, indicating no irritation to rabbit skin. In the eye irritation test, metsulfuron-methyl caused mild conjunctival redness and eyelid swelling, with an average irritation score of 0.325, classifying it as a mild irritant (Category 2B), while nicosulfuron and methoxyfenozide did not cause any observable eye irritation (score 0). The skin sensitization test revealed no erythema or edema reactions in guinea pigs exposed to the highest concentration (100%) of each pesticide, confirming that none of the samples caused skin sensitization. These findings provide essential toxicological data for regulatory safety evaluations, particularly for occupational and environmental exposure risks. Future research should explore chronic toxicity, long-term environmental impact, and transformation products of these pesticides, along with assessments in real-environment samples for more comprehensive safety insights.

Acknowledgments

We would like to thank the Rector of Universitas Padjadjaran for the Academic Leadership Grant (ALG) project No. 1408/UN6.3.1/PT00/2024 awarded to A.S. for funding the APC and PT. Sari Kresna Kimia for providing the sample.

Funding information: Authors state no funding involved.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal. All authors have read and agreed to the published version of the manuscript. Conceptualization and methodology: W.D.N., M.I.R., A.S., and G.W.; validation: G.W.; formal analysis: M.I.R. and G.W.; writing – original draft preparation: W.D.N. and M.I.R.; writing – review and editing: W.D.N., M.I.R., A.S., G.W., and C.S.; supervision, project administration, and funding acquisition: W.D.N., M.I.R., A.S., and G.W.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2025-01-20

Revised: 2025-03-21

Accepted: 2025-04-10

Published Online: 2025-05-06

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/opag-2025-0443

Keywords for this article

irritation test; sensitization test; metsulfuron-methyl; nicosulfuron; methoxyfenozide; toxicity

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