[No public or meaningful name is available]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Jan 2021 to 02 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

December 2001

Deviations:

yes

Remarks:

The standard dosing procedure for the starting dose of 300mg/kg in the Acute Toxic Class Method was not followed. The animals were dosed at 300 mg/kg, 2000 mg/kg and then 300 mg/kg.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Ec No 440/2008, part B: "Acute Oral Toxicity, Acute Toxic Class Method"

Version / remarks:

May 2008

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

December 2002

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines, including the most recent revisions

Version / remarks:

November 2000

Deviations:

not specified

GLP compliance:

yes

Remarks:

An exception from GLP was noted in the analysis of the test item in vehicle. It was determined that this did not affect the integrity of the study or the results.

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-3-methylbenzenesulphonic acid

Cas Number:

7134-04-5

Molecular formula:

C7H8O4S

IUPAC Name:

4-hydroxy-3-methylbenzenesulphonic acid

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Rationale for alternative/additional species to rat (if applicable): The rat was chosen as it is recognized by international guidelines as a recommended test system.
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Rationale for use of males (if applicable): NA
- Age at study initiation: 9-10 weeks old
- Weight at study initiation: 156g to 196g
- Fasting period before study: overnight
- Housing: Animals were group housed (3 animals in same dosing group together) in polycarbonate cages with sterilized wooden fibres as bedding and water bottles.
- Historical data: Not stated
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, municipal tap-water
- Acclimation period: 5 days
- Microbiological status when known: Not stated
- Method of randomisation in assigning animals to test and control groups: at the discretion of the coordinating biotechnician.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 44 to 60%
- Air changes (per hr): 10 or greater air changes with no recirculation
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark

IN-LIFE DATES: From: 06 January 2021 To: 02 February 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 300 mg/kg and 2000 mg/kg
- Amount of vehicle (if gavage): Not reported
- Justification for choice of vehicle: Water was chosen as the vehicle based on trial preparations, these results were not outlined in the study report
- Lot/batch no.: NA
- Purity: NA


CLASS METHOD
- Rationale for the selection of the starting dose: The dose levels were based on the OECD test guidelines and were selected from the series 5 (lowest dose level), 50, 300 and 2000 (highest dose level) mg/kg body weight. The starting dose level should be the one that is likely to produce mortality in at least some of the animals and was selected based on available toxicity data of the test item.

Doses:

The first group was treated at a dose level of 300 mg/kg. Based on the results, two additional groups were dosed at 2000 and 300 mg/kg respectively.

No. of animals per sex per dose:

3 females/group

Control animals:

no

Details on study design:

The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. The first group was treated at a dose level of 300 mg/kg. Based on the results, two additional groups were dosed at 2000 and 300 mg/kg.

Statistics:

No statistical analysis was performed.

Results and discussion

Preliminary study:

No signs of toxicity were noted following the initial dosing of 300 mg/kg, all animals were terminated on Day 15, following the experimental design a second group were dosed at 2000 mg/kg. All animals were found dead or sacrificed in moribund condition on Day 2 or Day 6 post-treatment. The rats exhibited shallow breathing, prostrate, erect fur, partly closed eyes and decreased activity were noted on Day 1 in both animals that were found dead on Day 2. the remaining animals were sacrificed in moribund condition on Day 6 with laboured breathing, hunched posture, erect fur, partly closed eyes, slight to moderate uncoordinated movements, abnormal gait decreased activity and pallor skin noted between Day 1 and 6. A third dosing of animals at 300 mg/kg showed signs of toxicity which included hunched posture and erect fur were noted in all animals between 1 and 3.

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

>= 300 - <= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

At 2000 mg/kg all animals were found dead or sacrificed in moribund condition. Two rats were found dead on day 2 with the third rat sacrificed after being found in a moribund condition on day 6.

Clinical signs:

other: At 2000 mg/kg shallow breathing, prostrate, hunched posture, erect fur, partly closed eyes and decreased activity were noted on Day 1 in both animals that were found dead on Day 2. The animal that was sacrificed in moribund condition on Day 6 showed labou

Body weight:

other body weight observations

Remarks:

The mean body weight gain shown by the surviving animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain, except for one animal at 300 mg/kg which showed slight body weight loss between Days 8 and 15.

Gross pathology:

At 2000 mg/kg the macroscopic post-mortem examination showed abnormalities of the stomach, which consisted of dark black discolouration of the glandular stomach, thick limiting ridge and glandular stomach, thin non-glandular stomach, a gritty black abnormal content and/or an irregular surface of the glandular stomach in the two animals found dead. In the animal that was sacrificed in moribund condition, decreased adipose tissue was observed.

At 300 mg/kg, no test item related alterations were seen at post mortem macroscopic examinations. One animal at 300 mg/kg showed a hernia of the diaphragm in the left medial lobe of the liver. This abnormality was considered to be a result of a birth defect and therefore was considered not to be test item related.

Any other information on results incl. tables

Table 1 Individual Mortality
Sex: Female Day(s): - Relative to Start Date

300 mg/kg Group 1	Day of death	Removal Date	Path Removal Reason
1	15	20-Jan-2021	TERM
2	15	20-Jan-2021	TERM
3	15	20-Jan-2021	TERM

 

2000 mg/kg Group 1	Day of death	Removal Date	Path Removal Reason
4	2	13-Jan-2021	FD
5	2	13-Jan-2021	FD
6	6	17-Jan-2021	UNSC

 

2000 mg/kg Group 1	Day of death	Removal Date	Path Removal Reason
7	15	02-Feb-2021	TERM
8	15	02-Feb-2021	TERM
9	15	02-Feb-2021	TERM

 

TERM = Terminal Euthanasia
FD = Found Dead
UNSC = Unscheduled Termination

 

Table 2: Individual Clinical Observations 

 

Group 1 Sex: Female 300 mg/kg
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
1	Hunched Posture	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
2	Hunched Posture	X	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erect	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
3	Hunched Posture	X	X	X	X	X
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Group 2 Sex: Female 2,000 mg/kg
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
4	Breathing, Shallow	.	.	X
	Prostrate	.	.	X
	Hunched Posture	X	X	X
	Fur, Erected	X	X	X
	Eye Closed, Left Partly	.	X	X
	Eye Closed, Right Partly	.	X	X
	Activity Decreased	.	X	X
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
5	Breathing, Shallow	.	.	X
	Prostrate	.	.	X
	Hunched Posture	X	X	X
	Fur, Erected	X	X	X
	Eye Closed, Left Partly	.	X	X
	Eye Closed, Right Partly	.	X	X
	Activity Decreased	.	X	X
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
6	Dehydrated Suspected Moderate	.	.	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.
	Breathing, Laboured	.	.	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.
	Hunched Posture	X	X	X	X	X	X	X	X	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	X	X	X	X	X	.	.	.	.	.	.	.	.	.
	Skin, Pallor	.	.	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.
	Eye Closed, Left Partly	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Eye Closed, Right Partly	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Thin	.	.	.	.	.	.	X	X	.	.	.	.	.	.	.	.	.
	Uncoordinated , Moderate	.	.	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.
	Uncoordinated , Slight	.	.	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Abnormal Gait	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.	.	.
	Activity Decreased	.	X	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.
Group 3 Sex: Female 300 mg/kg
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
7	Hunched Posture	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
8	Hunched Posture	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
9	Hunched Posture	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	.	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.
	Skin Lesion, w/discharge, Pinna, Right, Moderate	.	.	.	.	.	.	X
	Skin Lesion, w/discharge, Pinna, Right, Slight	.	.	.	.	.	.	.	X	X	X	X	.	.	.	.	.	.
	Skin Discoloured, Pinna, Right, Red	.	.	.	.	.	.	X	X	X	.	.	.	.	.	.	.	.
	Skin, Scab, Dorsal Cervical	X	X	X	X	X	X	X	X	.	.	.	.	.	.	.	.	.
	Swollen, Pinna, right, Moderate	.	.	.	.	.	.	X	.	.	.	.	.	.	.	.	.	.
	Swollen, Pinna Right, Slight	.	.	.	.	.	.	.	X	X	.	.	.	.	.	.	.	.

Group 1 Sex: Female 300 mg/kg
	Observation Type: All types	Day(s) Relative to start date
		1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
		0h	2h	4h
1	Hunched Posture	X	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
2	Hunched Posture	X	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.
3	Hunched Posture	X	X	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.
	Fur, Erected	X	X	X	.	.	.	.	.	.	.	.	.	.	.	.	.	.

X = Present 

. = Not Present 

 

Table 3: Individual Body weights

 

Sex: Female  

Bodyweight (g)

 

300 mg/kg Group 1	Day(s) Relative to Start date
	1	8	15
1	166	197	205
2	180	196	216
3	176	204	207
Mean	174.0	199.0	209.3
SD	7.2	4.4	5.9
N	3	3	3
2000 mg/kg Group 2	Day(s) Relative to Start date
	1
4	172*
5	157*
6	156**
Mean	161.7
SD	9.0
N	3
300 mg/kg Group 3	Day(s) Relative to Start date
	1	8	15
7	175	194	206
8	184	197	209
9	196	224	216
Mean	185.0	205.0	210.3
SD	10.5	16.5	5.1
N	3	3	3

 

 

* Animals found dead on Day 2. Terminal body weight: animal No. 4: 174, Animal No. 5: 149 grams

** Animal sacrificed in moribund condition on Day 6. Terminal body weight: 130 grams

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

LD50 cut-off value 500 mg/kg body weight

Conclusions:

Under the conditions of this study, the oral LD50 value was established within the range of 300-2000 mg/kg. According to the OECD TG 423 the LD50 cut-off was considered to be 500 mg/kg body weight, therefore the substance was classified as an Acute Oral Toxicity Category 4 according to the CLP Regulation (EC) No 1272/2008.

Executive summary:

The study was performed to OECD TG 423 under GLP to assess the acute toxicity potential of the test material when administered to female Wistar Hans rats at an initial dose of 300 mg/kg body weight. A dose volume of 10 mL/kg body weight was administered to three female fasted rats on day 1. In a stepwise procedure two additional groups of three female rats were dosed at 2000 and 300 mg/kg body weight. All animals were subjected to daily observations and weekly determination of body weight. All groups also underwent macroscopic examination performed on day of death or after terminal sacrifice. 

 

No mortality was observed in Group 1 300 mg/kg body weight, hunched posture and erected fur were noted in all animals between Days 1 and 3. No test item-related changes were noted at post-mortem macroscopic examination. Two animals were found dead in Group 2 2000 mg/kg body weight with the third female sacrificed on Day 6 post treatment. Clinical observations on day 1 were noted in both rats which were found dead on day 2, the observations included shallow breathing, prostrate, hunched posture, erected fur, partly closed eyes and decreased activity. The third female terminated on day 6 laboured breathing, hunched posture, erected fur, partly closed eyes, slight to moderate uncoordinated movements, abnormal gait, decreased activity and a pallor skin were noted between Days 1 and 6 with moderate dehydration was suspected on Day 6. Macroscopic examinations of the animals in Group 2 noted abnormalities of the stomach which consisted of dark black discoloration of the glandular stomach, a thick limiting ridge and glandular stomach, thin non-glandular stomach, gritty black abnormal content and/or and irregular surface of the glandular stomach in the two animals which were found dead on Day 2. In the third animal found dead on Day 6 a decrease in adipose tissue was observed.

 

No mortality was observed in Group 3 300 mg/kg body weight. All three animals were noted to have a hunched posture with erect fur between days 1 and 2. The third animal also showed a slight to moderately swollen and red discoloured right pinna skin lesion with slight to moderate discharge and a dorsal cervical scab between Days 1 and 9. This was not considered to be related to the test item, no test item-related changes were noted at post-mortem macroscopic examination.

 

The oral LD50 value of the test item was established within the range of 300-2000 mg/kg body weight. With the LD50 cut-off value was considered to be 500 mg/kg body weight. Based on the result the test item was classified as Acute Oral Toxicity Category 4 according to the CLP Regulation (EC) No 1272/2008.