Soybean oil, epoxidized, reaction products with methanol

Endpoint summary

Description of key information

In a GLP-conform study according to OECD test guideline 408, the administration of the test substance by gavage to male and female Wistar rats for 3 months caused no adverse treatment-related signs of toxicity up to and including the highest tested dose. Therefore, the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Sep 2021 - 04 Aug 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25 Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Batch No.: 0021987711
- CAS No.: 85586-35-2
- EC No.: 287-837-7
- Purity: 100 % UVCB (=Substances of unknown or variable composition, complex reaction products or biological material)
- Identity: Confirmed
- Homogeneity: Given (visually)
- Storage stability: Expiry date: 05 Jun 2022. The stability of the test substance under storage conditions over the test period was guaranteed by the Sponsor, and the Sponsor holds this responsibility.
- Physical state/ appearance: Liquid, viscous/ yellowish, clear
- Storage conditions: Room temperature

TEST-SUBSTANCE PREPARATIONS AND PREPARATION FREQUENCY
- The test substance was applied as an emulsion.
- To prepare this emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently mixed using a magnetic stirrer.
- The test substance preparations were produced at least once a week and stored at room temperature.
- During administration the test-substance preparations were kept homogeneous using a magnetic stirrer. The administration volume was 4 mL/kg body weight.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services
GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: mean 154 g (males) and 126 g (females)
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
- On basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable.
- On basis of the analytical findings the drinking water was found to be suitable.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00 h-18.00 h, 12 hours dark from 18.00 h-06.00 h

IN-LIFE DATES: From: 28 Sep 2021 To: 07 Jan 2022

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was applied as an emulsion.
- To prepare this emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently mixed using a magnetic stirrer.
- The test substance preparations were produced at least once a week and stored at room temperature.
- During administration the test-substance preparations were kept homogeneous using a magnetic stirrer.

ADMINISTRATION
- The administration volume was 4 mL/kg body weight.


VEHICLE: corn oil
- Concentration in vehicle: 0, 2.5, 7.5 and 25.0 g/100 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle, the correctness of the prepared concentrations in the test-substance preparations.

Duration of treatment / exposure:

3 month

Frequency of treatment:

daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the preliminary inlife results of an OECD 422 study (Project No.: 85R0114/12C188) the following dose levels were selected: 1000 mg/kg bw/da) as high concentration, 300 mg/kg bw/d as mid concentration and 100 mg/kg bw/d as low concentration.
- Fasting period before blood sampling for clinical biochemistry: yes, about 16 to 20 hours.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals.
- The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm height). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/ arousal level
8. Tremors
9. Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos
15.Assessment of the feces discharged during the examination (appearance/ consistency)
16.Assessment of the urine discharged during the examination (amount/color)
17.Pupil size


BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.


FOOD CONSUMPTION: Yes
- Food consumption was determined weekly (over 7 days) for each cage. T
- The average food consumption per cage was used to estimate the mean food consumption in grams per animal and day.


WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt
changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period, i.e. study day 90, the eyes of animals in test groups 0 (control) and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum, Bausch&Lomb GmbH, Berlin, Germany).


HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- The examinations were carried out in all surviving animals per test group and sex at the end of the administration period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- The examinations were carried out in all surviving animals per test group and sex at the end of the administration period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all:
- Parameters checked in table 3 were examined.

THYROID HORMONES: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- The examinations were carried out in all surviving animals per test group and sex at the end of the administration period.
- Animals fasted: Yes
- See table 4

URINALYSIS: Yes
- For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
- The examinations were carried out in all surviving animals per test group and sex at the end of the administration period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observational battery (FOB)
- Time schedule for examinations: A functional observational battery (FOB) was performed in all animals at the end of the administration period starting in the morning.
- At least one hour before the start of the FOB the animals were transferred to single-animal cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory-motoric tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Home cage observations: The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces (consistency/ color) excreted within two minutes
17. Urine (amount/ color) excreted within two minutes
18. Rearing within two minutes
19. Other findings
- Sensory-motoric tests/ reflexes: The animals were then removed from the open field and subjected to following sensory-motoric or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs and hindlimbs
13. Landing foot-splay test
- Motor activity assessment: Motor activity (MA) was also measured in the early afternoon onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

IMMUNOLOGY: No

ESTROUS CYCLE DETERMINATION: Yes
- For all females estrous cycle length and normality were evaluated by preparing vaginal smears during a minimum of 3 weeks prior to necropsy.
- Additionally, vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.

SPERM PARAMETERS: Yes
- After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male animals sacrificed on schedule.
- Parameters checked in table 6 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy: The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
- Organ weights: The following weights were determined in all animals sacrificed on schedule
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries (fixed)
9. Pituitary gland (fixed)
10. Prostate (ventral and dorsolateral part together, fixed)
11. Spleen
12. Seminal vesicles including coagulating glands (fixed)
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes (see table 8)
- Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
- Special attention was given for the male reproductive organs, especially the stage of seminiferous tubules.
- After completion of the histopathological assessment by the study pathologist an internal peer review was performed.

Statistics:

Means and standard deviations of each test group were calculated for several parameters (see tables). Further statistical analyses were performed according to following table 1,7 and 9.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In all male and all female animals of test group 3 (1000 mg/kg bw/d) slight to moderate salivation and the activity/ behavior of ploughing the nose-first into the bedding was observed within 2 hours after application on several days of the administration period starting on study day 5 and 6, respectively.
Five male animals (Nos. 21-23, 27 and 28) of test group 2 (300 mg/kg bw/d) showed slight salivation within 2 hours after application on several days of the administration period first seen on study day 26. Female animal No. 65 of test group 2 (300 mg/kg bw/d) showed slight salivation within 2 hours after application once on study day 84. All animals recovered from above mentioned clinical signs within 5 hours after application.
Animals of test groups 0 and 1 (0 and 100 mg/kg bw/d) were without any clinical signs. The slight to moderate salivation and the behavior of ploughing the nose-first into the bedding observed within 2 hours after application were considered to be treatment related due to a bad taste of the test-substance preparations administrated by gavage.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant statistically significantly changes of mean body weights and mean body weight change values in both sexes were observed.
In female animals of test group 2 (300 mg/kg bw/d) body weight change values were statistically significant increased on study days 14 and 42 in comparison to the control group. In females of test group 1 (100 mg/kg bw/d) body weight change was increased statistically significantly on study day 14 in comparison to the control group.Since these changes occurred at single time points and not dose dependend, they are assessed as incidental and not related to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test-substance related findings were observed.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test-substance related changes were observed.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings.
All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At the end of the administration period in males of test groups 1, 2 and 3 (100, 300, 1000 mg/kg bw/d) red blood cell (RBC) counts and hematocrit values were significantly increased. Changes in hematocrit values were not dose dependent. All values were within historical control ranges, apart from low RBC counts in the study controls below their range (males, RBC 8.03-8.86 Tera/L, hematocrit 0.406-0.438 L/L). Additionally in males of test group 3 (1000 mg/kg bw/d) platelet counts were significantly increased, but the values were within the historical control range (males, platelets 592-738 Giga/L). In females of test group 3 (1000 mg/kg bw/d) relative eosinophil and basophil counts were significantly decrease, but the values were also within historical control ranges (females, relative eosinophils 1.5-3.2 %, relative basophils 0.1-0.7 %). Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed. At the end of the administration period, in males of test group 3 (1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly increased. However, the increase was marginal (below 50%) compared to study controls. Because this is the only relevantly changed clinical chemistry parameter among these individuals, the alteration was regarded as maybe treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 3 (1000 mg/kg bw/d) LDL-cholesterol and triglyceride levels were significantly increased whereas sodium values were significantly decreased. Additionally, in males of test group 1 (100 mg/kg bw/d) triglyceride values were significantly increase, but this change was not dose dependent. All changed values were within historical control ranges (males, LDL-cholesterol 0.17-0.27 mmol/L, triglycerides 0.62-1.45 mmol/L, sodium 140.0-149.6 mmol/L). Therefore, these changes were regarded as incidental and not treatment related.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones:
No treatment-related alterations of T3, T4 and TSH levels were observed. In males of test group 3 (1000 mg/kg bw/d) T3 levels were significantly decreased, but the values were within historical control ranges (males, T3 0.75-1.35 nmol/L). Therefore, this change was regarded as incidental and not treatment related.

Spermanalysis:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.
At the end of the administration period in males and females of test group 3 (1000 mg/kg bw/d) as well as in females of test group 2 (300 mg/kg bw/d) urine volume was significantly decreased whereas specific gravity in the urine was significantly increased. Additionally, in males of test group 3 (1000 mg/kg bw/d) pH values were significantly decreased and in the urine sediment the incidence of granulated and epithelial casts was significantly increased. Low urine volume and high specific gravity in the urine reflect the adaptive capacity of the kidneys towards low fluid income. Low pH value of the urine in absence of any electrolyte change in the blood was most probably due to the excretion of acid metabolites of the administered compound. The higher incidence of casts in the urine of males of the age at the end of this study is most probably the consequence of alpha-2u globulinuria which is a typical finding of male rats of this age and is acknowledged as rat specific effect without human relevance. Therefore, the mentioned alterations in urine parameters were regarded as treatment related but nonadverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:

Home cage observations: No test-substance related adverse effects were observed.

Open field observations: No test-substance related adverse effects were observed.

Sensory-motoric test/ reflexes: No test-substance related adverse effects were observed. One male animal (No. 10) of test group 0 (0 mg/kg bw/d) showed an escape reaction during testing of approach response. This single finding was assessed as spontaneous in nature.

Quantitative parameters: No test-substance related adverse effects were observed.

Motor activity measurement: Regarding the overall motor activity as well as single intervals, no test-substance related deviations were noted for male and female rats.
The values of individual interval No. 11 were statistically significant decreased in male animals of test groups 1 and 3 (100 and 1000 mg/kg bw/d). The decrease of both values is a result of the unusual high value of male animals of the control group (0 mg/kg bw/d) and can be assessed as incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights:
When compared with control group 0 (=100%), the following mean absolute weights were significantly changed in one or more test groups (statistically significant change, see table 10). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.


Relative organ weights:
When compared with control group 0 (=100%), the following mean relative organ weights were significantly changed in one or more test groups (statistically significant changes, see table 11). All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The mean relative brain weight in females of test group 3 (1000 mg/kg bw/d) was statistically significantly increased. As the mean absolute weight of the brain in test group 3 (1000 mg/kg bw/d) females (1.998g) was not statistically significantly changed and was well within historical controls (1.91g - 2.017 g) this was considered secondary to the decreased terminal body weight of females of this group (-6.1%, not statistically significant). Comparing to historical controls, the mean relative weight in test group 3 (0.946%) was slightly above historical controls (0.84% - 0.91%). As there was no histopathological correlate in test group 3 (1000 mg/kg bw/d) and absolute weights were within historical controls, this was assessed as incidental.
The mean relative kidney weight in females of test group 3 (1000 mg/kg bw/d) was also statistically significantly increased. As the mean absolute weight of the kidneys in test group 3 (1000 mg/kg bw/d) females (1.539 g) was not statistically significantly changed and was well within historical controls (1.43g - 1.617g) this was considered secondary to the decreased terminal body weight of females of this group (-6.1%, not statistically significant). Comparing
to historical controls, the mean relative weight in test group 3 (0.728%) was, however, slightly above historical controls (0.64% - 0.71%). As there was no histopathological correlate in test group 3 (1000 mg/kg bw/d) and absolute weights were within historical controls, this was assessed as incidental.
The statistically significantly increased mean relative weights of the kidneys in male animals of test group 3 (1000 mg/kg bw/d) were assessed as incidental assumed to be caused by lower control weights (absolute 2.154 g, relative 0.552%) below historical controls (absolute 2.165 g - 2.41 g, relative 0.57% - 0.64%) while weights in test group 3 (1000 mg/kg bw/d) males (absolute 2.407g, relative 0.641%) were within historical controls (absolute 2.165g - 2.41g, relative 0.57 - 0.64%). Furthermore, there was neither a clear dose-response relationship nor a histopathological correlate in test group 3 (1000 mg/kg bw/d).
The statistically significantly increased mean absolute and relative liver weights in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) and the statistically significantly increased mean absolute and relative liver weights in males of test group 3 (1000 mg/kg bw/d) and the increased mean relative weight of the liver in test group 2 (300 mg/kg bw/d) males were assessed as treatment related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the jejunum of males and females of test group 3 (1000 mg/kg bw/d), and in liver and thyroid gland of males of test group 3 (1000 mg/kg bw/d).

Jejunum: In the jejunum of male and female animals of test group 3 (1000 mg/kg bw/d), minimal lymphangiectasis was observed. This was characterized by dilation of lymphatics in villi of the jejunum, the lymphatics were empty, and no tissue reaction was visible around them. Incidences are shown in the table 12.

Liver: In the liver of male animals of test group 3 (1000 mg/kg bw/d), minimal centrilobular hepatocellular hypertrophy was noted with incidences according to the table 13.

Thyroid glands: In the thyroid glands of male animals of test group 3 (1000 mg/kg bw/d), minimal hypertrophy/hyperplasia of follicular cells was observed with minimally increased incidence over controls, which was considered equivocally treatment related. Altered colloid, characterized by flaky basophilic appearance was noted in one control and two animals each of test group 2 and 3 (300 and 1000 mg/kg bw/d). Incidences and grading of these findings are shown in the table 14.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle
No statistically significant test-substance related effects on estrous cycle length and the number of cycles were obtained in female animals of all test groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to the highest tested dose.

Critical effects observed:

no

Tab. 10: Absolute organ weights

 

		Male animals			Female animals
Test group	1		2	3	1	2	3
(mg/kg bw/day)	(100)		(300)	(1000)	(100)	(300)	(1000)
Final body weight					+1.7%	+1.7%	-6.1%
Liver	+4.8%		+6.7%	+16.1%*	-0.9%	+9.8%*	+6.1%*

*: p <= 0.05, **: p <= 0.01

 

 

Tab. 11: Absolute organ weights

 

	Male animals			Female animals
Test group	1	2	3	1	2	3
(mg/kg bw/day)	(100)	(300)	(1000)	(100)	(300)	(1000)
Brain				-1.6%	-1.1%	+7.1%
Kidneys	+7.7%	+4.8%	+16.1%**	-4.9%	+0.7%	+10.0%
Liver	+4.0%	+5.7%*	+20.5%**	-2.3%	+7.9%	+13.0%

*: p <= 0.05, **: p <= 0.01

 

 

Tab. 12: Incidences of jejunum findings

 

	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (100)	2 (300)	3 (1000)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10	10	10	10	10
Lymphangiectasis	0	0	0	3	0	0	0	6
·         Grade 1				3				6

 

 

Tab. 13: Incidences of liver findings

 

	Male animals
Test group (mg/kg bw/day)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10
Hypertrophy, centrilobular	0	0	0	5
·         Grade 1				5

 

 

Tab. 14: Incidences of thyroid glands findings

 

	Male animals
Test group (mg/kg bw/day)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10
Hypertrophy/hyperplasia, follicular cell	1	0	0	3
·         Grade 1	1			3
Alteration, colloid	1	0	2	2
·         Grade 1	1		2	2

 

Conclusions:

In a GLP-conform study according to OECD test guideline 408, the administration of the test substance by gavage to male and female Wistar rats for 3 months caused no adverse signs of toxicity up to and including the highest tested dose.

Therefore, the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats.

Executive summary:

In a GLP-conform study according to OECD test guideline 408, the test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (test group 3) over a period of 3 months.

 

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs before and within 2 hours as well as within 5 hours after treatment. Moreover, detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.

Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

 

The various analyses confirmed:

• the stability of the test-substance preparations for a period of 7 days at room temperature,


• the homogeneous distribution of the test substance in the vehicle,


• the correctness of the prepared concentrations in the test-substance preparations.

The administration of the test substance by gavage to male and female Wistar rats for 3 months caused no adverse signs of toxicity up to and including the highest tested dose.

Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose 90-Day Oral Toxicity Study in Rodents:

In a GLP-conform study according to OECD test guideline 408, the test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 100 (test group 1), 300 (test group 2) and 1000 mg/kg body weight/day (test group 3) over a period of 3 months.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. In addition, the rats were daily examined for any clinically abnormal signs before and within 2 hours as well as within 5 hours after treatment. Moreover, detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.

Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

The various analyses confirmed the stability of the test-substance preparations for a period of 7 days at room temperature, the homogeneous distribution of the test substance in the vehicle, and the correctness of the prepared concentrations in the test-substance preparations.

The administration of the test substance by gavage to male and female Wistar rats for 3 months caused no adverse treatment-related signs of toxicity up to and including the highest tested dose. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d for male and female Wistar rats. (BASF 2022)

 

 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test:

In an OECD 422 study, CAS 85586-35-2 was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (corn oil). The duration of treatment covered a 28 days in-life period in males (including premating, mating [mating pairs were from the same test group] and postmating period) and a 2-weeks premating and mating period, the entire gestation and approximately 3 weeks of lactation period in females. Parental females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or 13. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions. Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. Regarding clinical examinations, in females no test substance related, adverse signs of (systemic) toxicity were observed up to limit dose of 1000 mg/ kg bw/d. All males and females of the high-dose (1000 mg/kg bw/d) and two out of ten males of the middose (300 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is most likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. In male body weight development, a slight overall downward trend of the high dose group may indicate the beginning of systemic toxicity. This reduction was small (about 8% body weight below control values) and only a borderline case; however, as toxicity cannot be excluded, it is considered as adverse. Neither water nor food consumption were adversely affected in any of the tested dose groups. Regarding clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, target organs were the kidneys and the liver in males of test group 3. As the increase of the mean relative (+9.4%) and absolute (+19.0%) kidney weight as well as of the mean relative liver weight (+15.0%) was not accompanied by histopathological changes, these findings were regarded as possibly treatment-related, but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. In conclusion, the oral administration of CAS 85586-35-2 by gavage to female Wistar rats resulted in no adverse signs of systemic toxicity up to limit dose of 1000 mg/kg bw/d. In male rats the NOAEL for general, systemic toxicity of the test substance is 300 mg/kg bw/d based on the changes to body weight parameters at 1000 mg/kg bw/day. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d. (BASF 2022)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.