6-methyl-2-oxoperhydropyrimidin-4-ylurea

Endpoint summary

Description of key information

Oral (OECD 422, RA from CAS 6104-30-9), rat: NOAEL=1000 mg/kg bw/day (systemic, male/female); (similar to OECD 416/421, RA from CAS 6104-30-9), rat: NOAEL=1200 mg/kg bw/day (systemic, male/female)
Inhalation: No data available.
Dermal: No data available.

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted similar to the OECD test guidelines 416 and 421, and in compliance with GLP. Since this is a read-across from a structural analogue substance (CAS 6104-30-9), the reliability was set from RL1 to RL2.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

(the study focused on fertility/reproduction only and there was no 2nd generation)

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

(the study focused on fertility/reproduction only and there was no 2nd generation)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle, France
- Age at study initiation: (P) 6 weeks
- Weight at study initiation: (P) Males: 171-210 g (mean: 192 g); females: 142-179 g (mean: 161 g)
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage. The cages were placed in numerical order on the racks. On a monthly basis, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a 6-day acclimation period to the conditions of the study preceded the beginning of the treatment period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 °C
- Humidity (%): 50±20 %
- Air changes (per hr): about 12 cycles filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 101K0185, supplied by Sigma (Saint-Quentin-Fallavier, France).
- The test substance was administered as a suspension in the vehicle.
- The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 60 and 120 mg/mL and then homogenised using an Ultraturrax laboratory mixer pending 5 minutes and a magnetic stirrer.
- The test substance dosage forms were made daily (preparation stable for 3 hours at room temperature).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity were determined on samples of each control and test item dosage form prepared for use in week 1, 7 and 13. Three samples of each dosage form (from top, middle and bottom of the container) were taken on each occasion. They were stored at -20 °C pending dispatch (two consignments). The samples were sent on dry ice to the Sponsor. These analyses were carried out under the responsibility of the Sponsor.

Duration of treatment / exposure:

- males: throughout the pre-mating period (10 weeks), mating period (2 weeks), and until sacrifice;
- females: throughout the pre-mating period (10 weeks), mating period (2 weeks), and pregnancy until day 14 post-coitum inclusive.

Frequency of treatment:

daily 7 days per week

Remarks:

Doses / Concentrations:
600 and 1200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were specified by the Sponsor and based on the available toxicological data obtained in a combined repeat dose toxicity/developmental toxicity screening test with the test item: 600 mg/kg bw/day as the expected No Adverse Effect Level, 1200 mg/kg bw/day as themaximum feasible dose-level.
The oral route was selected since it is a possible route of exposure in human and it is requested by the regulatory authorities for this type of test item.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded once a week until sacrifice. The body weight of each female was recorded once a week during the pre-mating and mating periods, then on days 0, 7 and 15 post-coitum.

Sacrifice and pathology:

SACRIFICE
- males: all surviving animals after most hysterectomies of the females had been performed
- maternal animals: all surviving animals on Day 15 post coitum
- females which did not mate: at least one week after the end of the mating period

GROSS NECROPSY
- Gross necropsy consisted of a macroscopic post-mortem examination of the principal thoracic and abdominal organs (with particular attention paid to the reproductive organs) was performed on all animals, including those that died during the study or were killed prematurely. For all females, the number of corpora lutea and implantation sites was recorded whenever possible. Whenever necessary, photographs were taken to document findings and kept with the study archives.

HISTOPATHOLOGY / ORGAN WEIGHTS
No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment. The body weight of all animals killed at terminal sacrifice was recorded and the following organs were weighed (wet) as soon as possible after dissection: all males: testes, epididymides, prostate, seminal vesicles together with coagulating glands, pituitary gland and adrenals, testes and epididymides were weighed separately, all females: uterus, ovaries, pituitary gland and adrenals.

Other examinations:

- reproductive indices: mating data, male fertility data, female fertility data (female fertility and gestation indices, hysterectomy parameters)
- sperm parameters examined in [P] male parental generations: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, and sperm morphology

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances
being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statisticallly significantly reduced in dams at 600 and 1200 mg/kg bw/day, no effect in males and in females before pregnancy. This effect is secondary to decreased food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statisticallly significantly reduced in dams at 1200 mg/kg bw/day, no effect in males and females before pregnancy. This is considered to be based on abnormal taste and smell perception during pregnancy and as protective mechanism.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment.

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY (PARENTAL ANIMALS)
- Males: No mortality was recorded in the control or the 1200 mg/kg/day groups. One male given 600 mg/kg/day was found dead on day 44. At macroscopic post-mortem examination, esophagus perforation was noted together with serous contents in the thoracic cavity and whitish deposit on the pleura. This death was considered of accidental nature and without any relationship to treatment with the test item.
- Females: There was no death, in any control or treated group over the treatment period.

CLINICAL SIGNS (PARENTAL ANIMALS)
- Males: The few clinical signs recorded in three males given 600 mg/kg/day (piloerection or chromodacryorrhea and chromorhinorrhea) and one male given 1200 mg/kg/day (ptyalism) were not considered to be of toxicological importance since the incidence of these findings was low and not dose-related.
- Females: The few clinical signs (ptyalism) recorded in one female given 600 mg/kg/day and three females given 1200 mg/kg/day did not represent an adverse effect. Other observations (area of hair loss on the forelimb or hindlimb) recorded in some males and females are among findings commonly observed in rats of this strain and age.

FOOD CONSUMPTION AND BODY WEIGHT (PARENTAL ANIMALS)
- Males: The food consumption of the treated males was similar to that of the control group.
- Females: During pre-mating (days 1 to 71), the food consumption was similar in the control and the treated groups. During pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively.

- Males: There was a minimal non significant decrease in body weight gain in males given 600 (-6%) and 1200 mg/kg/day (-5%) over the treatment period (days 1 to 92). Because these minor changes in body weight gain were not statistically different, not dose-related and did not correlate with any change in food consumption, they were considered to be marginal and of no toxicological significance.
- Females: During the pre-mating period (days 1 to 71), the body weight gain at 600 mg/kg/day was similar to that of the controls, while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption were considered to be not adverse, since the effect was observed in pregnant females solely and did not affect the reproductive outcome.

Expert judgement:
These changes on body weight gain and food consumption were considered to be not adverse, since the effect was observed in females solely, and not before pregnancy. It is generally known, that abnormal taste and smell perception is observed during pregnancy. It can therefore be expected that reduced food consumption is a consequence of abnormal taste and smell during gestation and may be a foeto-protective mechanism to avoid poisoning. This protective mechanism is expected to be enhanced by treatment with the test item, as a dose relationship was observed. In conclusion, the decreased body weight gain during pregnancy is considered to be more a gestation-related effect rather than a test-material related effect. Moreover, the decreased body weights did not affect the offspring, thus supporting that this observation can be concluded as non-adverse.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The minimal fluctuations recorded in the seminology parameters in the treated groups were not dose-related, not notably different from the controls, and in the range of CIT historical control data. Consequently, they were not considered of toxicological significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with their respective controls, higher absolute and relative adrenal weights were observed for treated males and lower absolute and relative ovary and uterus weights were noted for treated females. Considering that the differences in the adrenal weights were not dose-related and observed in the males only, they were considered to be without relationship to the treatment.
The differences in the weights of the reproductive organs, particulary for uterus and ovaries were considered not to be treatment-related. Indeed, most of individual values of ovaries weight at 600 and 1200 mg/kg/day were within the range of control group values. Concerning the slight decrease observed in uterus weight, this latter change was only due to the contribution of a few individual values of two females. In addition, the differences (% from control group) in the ratio of uterus weight/number of foetuses and of ovaries weight/number of corpora lutea between the treated and control animals was minor and not dose-related. In conclusion, these minor differences from the control group in ovaries and uterus weights were considered not to be related to the treatment with the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic findings recorded were those, which are commonly observed spontaneously in the untreated laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
MORTALITY (PARENTAL ANIMALS)
- Males: No mortality was recorded in the control or the 1200 mg/kg/day groups. One male given 600 mg/kg/day was found dead on day 44. At macroscopic post-mortem examination, esophagus perforation was noted together with serous contents in the thoracic cavity and whitish deposit on the pleura. This death was considered of accidental nature and without any relationship to treatment with the test item.
- Females: There was no death, in any control or treated group over the treatment period.

CLINICAL SIGNS (PARENTAL ANIMALS)
- Males: The few clinical signs recorded in three males given 600 mg/kg/day (piloerection or chromodacryorrhea and chromorhinorrhea) and one male given 1200 mg/kg/day (ptyalism) were not considered to be of toxicological importance since the incidence of these findings was low and not dose-related.
- Females: The few clinical signs (ptyalism) recorded in one female given 600 mg/kg/day and three females given 1200 mg/kg/day did not represent an adverse effect. Other observations (area of hair loss on the forelimb or hindlimb) recorded in some males and females are among findings commonly observed in rats of this strain and age.

FOOD CONSUMPTION AND BODY WEIGHT (PARENTAL ANIMALS)
- Males: The food consumption of the treated males was similar to that of the control group.
- Females: During pre-mating (days 1 to 71), the food consumption was similar in the control and the treated groups. During pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively.

- Males: There was a minimal non significant decrease in body weight gain in males given 600 (-6%) and 1200 mg/kg/day (-5%) over the treatment period (days 1 to 92). Because these minor changes in body weight gain were not statistically different, not dose-related and did not correlate with any change in food consumption, they were considered to be marginal and of no toxicological significance.
- Females: During the pre-mating period (days 1 to 71), the body weight gain at 600 mg/kg/day was similar to that of the controls, while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption were considered to be related to treatment with the test item.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The minimal fluctuations recorded in the seminology parameters in the treated groups were not dose-related, not notably different from the controls, and in the range of CIT historical control data. Consequently, they were not considered of toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The female mating index (mated/paired) and pre-coital interval were similar in all groups. Most paired animals mated within 1 to 4 days of cohabitation except for a few pairs in each group, for which the duration was slightly longer. This finding, commonly recorded at this low incidence was not attributed to treatment with the test item. The slight fluctuations in male fertility parameters, which were not dose-related, are commonly recorded in rats of this strain and age, consequently, they were not considered to be related to the treatment with the test item.
The treatment with the test item did not disturb fertility and gestation indices of the females at any dose-level. The number of corpora lutea was minimally lower in the treated groups when compared to the controls. Since the difference was small, not statistically significant, and within the range of CIT historical control data, these findings were considered to have occurred by chance. Hence, at 600 and 1200 mg/kg/day the number of implantation sites and concepti was also lower than the control, gaining statistical significance only at-the top dose-level. Since these values were also within CIT historical control data, they were considered to be a consequence of the fortuitous lower number of corpora lutea rather than a test item related effect.
The slight fluctuations in the pre- and post-implantation losses were not considered to be related to the treatment with the test item since they were not dose-related and/or not significantly different from either the concurrent control or the CIT historical control data.
ORGAN WEIGHTS (PARENTAL ANIMALS)
When compared with their respective controls, higher absolute and relative adrenal weights were observed for treated males and lower absolute and relative ovary and uterus weights were noted for treated females. Considering that the differences in the adrenal weights were not dose-related and observed in the males only, they were considered to be without relationship to the treatment.
The differences in the weights of the reproductive organs, particulary for uterus and ovaries were considered not to be treatment-related. Indeed, most of individual values of ovaries weight at 600 and 1200 mg/kg/day were within the range of control group values. Concerning the slight decrease observed in uterus weight, this latter change was only due to the contribution of a few individual values of two females. In addition, the differences (% from control group) in the ratio of uterus weight/number of foetuses and of ovaries weight/number of corpora lutea between the treated and control animals was minor and not dose-related. In conclusion, these minor differences from the control group in ovaries and uterus weights were considered not to be related to the treatment with the test item.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The few macroscopic findings recorded were those, which are commonly observed spontaneously in the untreated laboratory rat of this strain and age and thus considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment.

Dose descriptor:

NOAEL

Effect level:

1 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced body weight gain

Critical effects observed:

not specified

Additional data:

ORGAN WEIGHTS:

- females: significantly lower absolute and relative ovary and uterus weights (ovaries: low, high-dose/absolute: -12, -13%; low, high-dose/relative: -7, -4% as compared to controls; uterus: low, high-dose/absolute: -19, -21%; low, high-dose/relative: -14, -12% as compared to controls). These differences were considered not to be treatment related, as most of the values were within the range of the control group, and changes were only due to the contribution of a few individual values of two animals.

- males: significantly, but not dose-related higher absolute and relative adrenal weights (low, high-dose/absolute: +24, +18%; low, high-dose/relative: +29, +23% as compared to controls). Because of the lack of a dose-response and because only seen in males, these effects were considered as of no biological significance.

 

MATING DATA:

The female mating index (mated/paired) and pre-coital interval were similar in all groups (control, low, high-dose: female mating index 96 - 96 - 96%, mean pre-coital interval 2.7 - 2.8 - 3.2 days).

 

MALE FERTILITY DATA:

The male mating index and the male fertility index were similar in all groups (male mating index: control, low, high-dose: 100 - 96 - 96 %, male fertility index: 100 - 91.3 - 91.3 %).

 

FEMALE FERTILITY DATA:

The following was obtained for the control, low, and high-dose groups, respectively:

- mated females: 24, 24; 24

- pregnant females: 24, 21, 22

- female fertility index (%): 100, 87.5, 91.7

- females with live concepti: 24, 21, 22

- gestation index (%): 100, 100, 100

 

NUMBER OF CORPORA LUTEA, NUMBER OF IMPLANTATIONS (control, low, high-dose groups):

- corpora lutea: 17.6, 16.4, 16.0

- implantation sites: 16.6, 14.9, 14.5* (p<0.05)

- pre-implantation loss (%): 5.7, 9.6, 9.1

- concepti: 15.2, 14.3, 13.0* (p<0.05)

- post-implantation loss (%): 8.3, 4.2, 11.0

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

The female mating index (mated/paired) and pre-coital interval were similar in all groups. Most paired animals mated within 1 to 4 days of cohabitation except for a few pairs in each group, for which the duration was slightly longer. This finding, commonly recorded at this low incidence was not attributed to treatment with the test item. The slight fluctuations in male fertility parameters, which were not dose-related, are commonly recorded in rats of this strain and age, consequently, they were not considered to be related to the treatment with the test item.

The treatment with the test item did not disturb fertility and gestation indices of the females at any dose-level. The number of corpora lutea was minimally lower in the treated groups when compared to the controls. Since the difference was small, not statistically significant, and within the range of CIT historical control data, these findings were considered to have occurred by chance. Hence, at 600 and 1200 mg/kg/day the number of implantation sites and concepti was also lower than the control, gaining statistical significance only at-the top dose-level. Since these values were also within CIT historical control data, they were considered to be a consequence of the fortuitous lower number of corpora lutea rather than a test item related effect.

The slight fluctuations in the pre- and post-implantation losses were not considered to be related to the treatment with the test item since they were not dose-related and/or not significantly different from either the concurrent control or the CIT historical control data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional groups, common breakdown products, and similarities in PC/ECOTOX/TOX properties (refer to endpoint discussion for further details).

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

Justification for read-across

No sufficient data are available for the repeated dose toxicity of 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6). In order to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Repeated dose toxicty

CAS	1129-42-6 TARGET SUBSTANCE	6104-30-9
Chemical Name	6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur)	N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur)
MW	172.1851 g/mol	174.2010 g/mol
Repeated dose toxicity oral	RA: CAS 6104-30-9	NOAEL ≥ 1200 mg/kg bw/day

 

The above mentioned substances are considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

 

Repeated dose toxicity oral

No data are available for 6-methyl-2-oxoperhydropyrimidin-4-ylurea (Crotodur, CAS 1129-42-6), however, 2 reliable studies are available for the structurally analogue substance N,N”-(2-methylpropane-1,1-diyl) diurea (Isodur, CAS 6104-30-9), which were used for read-across based on the analogue approach.

In the available key study the structural analogue substance CAS 6104-30-9 was investigated for toxicity after repeated oral exposure (CIT, 2003a). The study was conducted according to OECD 422, and in compliance with GLP. 10 Sprague-Dawley rats per sex per dose received the test item in carboxymethyl cellulose via gavage at doses of 100, 300, and 1000 mg/kg bw/day. Males were treated throughout pre-mating (15 days), and during the mating and post-mating periods until sacrifice (34 days in total). Females received the test material throughout pre-mating (15 days) and mating period, during pregnancy and lactation, and until day 4 post partum. Treatment was conducted once daily for 7 days per week; concurrent controls received the vehicle only. No substance related deaths and no substance related clinical signs were observed throughout the study period in neither males nor females. Body weight gain was normal in males up to the limit dose and in females in the low and mid dose groups. High dose group females (1000 mg/kg bw/day) showed lower body weight gain during gestation (-10% on days 0-20 of pregnancy as compared to the controls), and during lactation (-38% on days 1-4 post-partum) in combination with slightly lower food consumption during gestation (-6% on days 0-20 of pregnancy). Since the reduced body weight gain, as a result of decreased food consumption, was not observed in males and only in females, as soon as they were pregnant, this effect was considered to be not toxicologically relevant. It is further generally known from the literature, that abnormal taste and smell perception is observed during pregnancy, and was reported not only for rodents (Clarke, S. N. & Bernstein, I. L., 2001), but is also widely known for humans (Klimack-Nawrot, E., et al., 2012; Nordin, S., et al., 2004; Poothullil, J.M., 1995). It can therefore be expected that reduced food consumption is a consequence of abnormal taste and smell during gestation and may be a foeto-protective mechanism to avoid poisoning (Nordin, S., et al., 2004). This protective mechanism is expected to be enhanced by treatment with the test item, as a dose relationship was observed. The fact, that the body weight gain was still reduced during lactation, is considered to be a secondary effect, since it is known that during lactation mammals have high energy needs. It is concluded that this is why the body weights did not normalise during lactation, although food consumption was comparable to the controls again. In conclusion, the decreased body weight gain during pregnancy is considered to be more a gestation-related effect rather than a test-material related effect. Moreover, the decreased body weights did not affect the offspring, thus supporting that this observation can be concluded as non-adverse.

Clinical chemistry examination revealed for females increased alanine aminotransferase at the top-dose of 1000 mg/kg bw/day (18 as compared to 10 in controls; p<0.01) and a slight increase of proteins in mid-dose females (300 mg/kg bw/day). Since the values were within historical control range, the effects were not considered as of biological significance. Males showed significant, but not dose-related slight increases in Na, Cl, and aspartate aminotransferase values. All values were within historical control ranges and therefore not considered as of biological significance. No effects were observed on haematology, urinalysis, and neurobehaviour for neither males nor females in any dose group. At gross pathology, no substance related pathological changes were noted and no treatment related effects on organ weights were reported. At histopathology, a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the 300 and 1000 mg/kg bw/day group males was noted. As no tubular degeneration/necrosis was observed in the kidneys the presence of acidophilic globules was considered as due to the accumulation of the sex-linked alpha-2-µ-globulin. This was confirmed by specific immunostaining in a specific investigation. As a sex- and species-specific effect of male rats, this finding has no relevance for humans. It was therefore considered as of minor toxicological importance and it was not considered as an adverse effect. The seminiferous tubules were lined with Sertoli cells only (minimal or slight) in 1/10 males given 300 mg/kg/day and in 2/10 males given 1000 mg/kg/day. For a third male from the same group, tubules lined with Sertoli cells only were considered to be tubuli recti as they were situated beneath the capsule. For 1/10 males given 300 mg/kg/day and another given 1000 mg/kg/day, minimal reduction in the number of spermatids was observed in very few seminiferous tubules. Minimal vacuolisation of Sertoli cells was observed in 1/10 males given 1000 mg/kg/day. Although not found in the control males, these microscopic abnormalities recorded with minimal severity in few or very few seminiferous tubules in a few males were considered to be without relationship to the treatment and most probably fortuitous. In summary, no treatment-related abnormalities were found in testes, epididymides, prostate, seminal/vesicles, ovaries, and uterus, and in all other investigated organs. In conclusion, the NOAEL deduced for systemic toxicity for both males and females was 1000 mg/kg bw/day due to toxicological unimportance of the effects observed.

Further information from the structural analogue substance CAS 6104-30-9 was obtained from a study actually performed to investigate the toxicity to reproduction (CIT, 2003b). The study was conducted similar to the OECD test guidelines 416 and 421, and in compliance with GLP. 25 Sprague-Dawley rats per sex per dose received the test substance in carboxymethyl cellulose via gavage at doses of 600 mg/kg bw/day and 1200 mg/kg bw/day. Males were treated throughout the pre-mating period (10 weeks), mating period (2 weeks), and until sacrifice; the females received the test material throughout the pre-mating period (10 weeks), mating period (2 weeks), and pregnancy until day 14 post-coitum inclusive. Treatment was carried out daily on 7 days per week, and concurrent controls received the vehicle only. Cage side observations were conducted at least twice a day, and detailed clinical observations were performed at least once daily. Body weights of each male were recorded once a week until sacrifice, and of each female once a week during the pre-mating and mating periods, then on days 0, 7 and 15 post-coitum. At study termination, the animals were sacrificed and submitted to macroscopic examination and organ weight evaluation. No microscopic examination was deemed necessary since all the macroscopic lesions were considered to be unrelated to the treatment. The study did not reveal any treatment-related effects on neither systemic toxicity nor reproductive function. Food consumption was similar for controls and treated animals during pre-mating (days 1 to 71). However, during pregnancy (days 1 to 15), minimal (-7%) and slight (-14%, p<0.01) decreases in food consumption were recorded at the 600 and 1200 mg/kg/day dose-levels, respectively. The body weight gain at 600 mg/kg/day was similar to that of the controls during the pre-mating period (days 1 to 71), while it was slightly lower (-11 %, p<0.05) in females given 1200 mg/kg/day. During pregnancy (days 1 to 15), a similar moderate decrease in body weight gain was recorded in females given 600 (-24%, p<0.001) and 1200 mg/kg/day (-24%, p<0.001). These changes on body weight gain and food consumption were not considered to be adverse. Similarly to the results of the screening study (CIT, 2003a), the effect was observed in pregnant females solely. Comparing the outcome of both the screening study and the one-generation study emphasises that the reduced food consumption and decreased body weight gain seem to be related to pregnancy, since this effect was not observed during the pre-mating period of 2 weeks (CIT, 2003a) or 10 weeks (CIT, 2003b). This emphasises that the dams did not reduce food consumption until gestation, independent of the exposure duration beforehand, further supporting that this is associated with abnormal taste and smell perception during pregnancy as described above. As complete recovery of the dams is expected after partuition and the F1 generation was not affected, the observed effect is considered to be temporary and thus of no toxicological relevance for systemic toxicity after repeated exposure. Based on these results, the NOAEL for systemic toxicity was set at 1200 mg/kg bw /day for both males and females.

In summary, no systemic toxicity was observed in neither the screening study with subacute exposure duration, nor in the one-generation study with subchronic treatment before mating. No signs of specific target organ toxicity were noted in any of the studies except for sex-linked alpha-2-µ-globulin accumulation in the kidneys of treated males. However, this effect is known to be of no toxicological relevance for humans. Thus, the NOAEL of ≥1200 mg/kg bw/day deduced from the one-generation study with subchronic treatment is considered to be reliable and therefore further testing for toxicity after repeated exposure is scientifically not justified.

References:

Clarke, S. N. & Berrnstein, I. L. (2001). NaCL increases during pregnancy and lactation: assessment using brief access tests. Pharmacol Biochem Behav. 68(3): 555-563

Klimacka-Nawrot, E., Suchecka, W., Hartmann, M., Gałazka, A., Musialik, J., Petelenz, M. (2012). Changes id food preferences in pregnant women. Wiad Lek. 65(1): 10-14

Nordin, S., Broman, D. A., Olofsson, J. K., Wulff, M. (2004). A longitudinal Descriptive Study of Self-reported Abnormal Smell and Taste Perception in Pregnant Women. Chem. Senses 29: 391-402

Poothullil, J. M. (1995). Neurosci Biobehav Rev. 19(3): 407-412

 

 

Repeated dose toxicity inhalation

No data available.

 

Repeated dose toxicity dermal

No data available.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Reliable data are available for the oral route.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Reliable data are available for the oral route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Reliable data are available for the oral route.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Reliable data are available for the oral route.

Justification for classification or non-classification

Based on read-across from the structurally similar substance, the available data on oral repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.