Urea, reaction products with formaldehyde, glyoxal and methanol

Endpoint summary

Description of key information

DMDHEU (a surrogate of the submission substance) was subjected to test subchronic oral toxicity in male and female rats and mice. In this study a NOAEL of 3000 mg/kg bw/day was identified for rats (based on statistically significant reduction of male bw compared to controls). There were no adverse effects found in mice, the resulting NOAEL = 6000 mg/kg bw/day (i.e. highest dose tested). Moreover the sumission substance was subjected to a combined repeated dose toxicity study with reproduction/developmental toxicity screening test using male and female rats. Oral dosing for at least 28 days (males, females were treated up to approx. 7 weeks), didn't reveal any treatment-related findings on parental animals, thus resulting in a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed database.

Principles of method if other than guideline:

Performed according to the NTP protocol

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Ninety three male and 96 female Fischer 344 rats were acclimated for a period of 15 days before treatment. Based on daily observations and a pre-initiation health verification conducted one week prior to study initiation, 90 males and 92 females were selected for possible use on study. A gross necropsy was performed on five males and 5 females one day prior to test initiation. All rats examined were found to be disease and parasite-free. Animals were randomly allocated to 4 groups of 10 animals/sex. They were 44-51 days old at study initiation. Males weighed 104-161 g and females weighed 96-115 g. Food and water were available ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Doses were given at a volume of 20 ml/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative solutions were analyzed immediately after preparation at two labs. A sample taken from the 1000 mg/kg dosage level solution was analyzed to be 110% of the target concentration at one lab and 97% of the target concentration at the other lab.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

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

Remarks:

(referring to 100 % substance)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

(referring to 100 % substance)

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

Remarks:

(referring to 100 % substance)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: no

Observations and examinations performed and frequency:

Animals were weighed prior to dosing, at weekly intervals, and at termination. Animals were observed daily for mortality and clinical signs of toxicity.

Sacrifice and pathology:

All survivors were euthanized on day 91 and received complete post-mortem examinations. The brain, lung, heart, thymus, liver, right testis and right kidney were weighed and relative weights (to brain and body) were calculated. These tissues, plus the pituitary, eyes, nasal cavity and turbinates, oral cavity, larynx and pharynx, tongue, salivary gland, Zymbal's gland, trachea, thyroid, parathyroid, mandibular lymph node, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, sternum (with marrow), aorta, costochondral junction (rib), spinal cord, mammary gland, skin, mesenteric lymph node, pancreas, spleen, adrenal, urinary bladder, testes with epididymis, tunica of the testis and scrotal sac, seminal vesicles, prostate, ovary, uterus, preputial or clitoral gland, thigh muscle, blood smear, sciatic nerve, gross lesions, tissue masses or suspect tumors and regional lymph nodes were saved in neutral 10% formalin. Stained sections of all collected organs from the high dose and control animals (except the tongue, Zymbal's gland, costochondral junction (rib), skin, seminal vesicles, thigh muscle and sciatic nerve) were examined histologically. The eyes and pharynx were only examined if grossly abnormal. Histologic sections of heart and testis were examined for the low and mid-dose males.

Other examinations:

At study termination, serum samples from 5 rats/sex from the control groups were analyzed for the presence of antibodies to murine viruses designated by the NTP. Positive titers to both Sendai (all rats) and PVM virus (all females) were detected using a hemagglutination inhibition assay.

Statistics:

Body and organ weight data of treated animals were compared to controls using a one-way analysis of variance, Bartlett's test for homogeneity of variances and the appropriate t-test (for unequal and equal variances) as described by Steel and Torrie. Dunnett's multiple comparison tables were used to assess significant differences at p < 0.05.

Details on results:

Three males of the 6000 mg/kg/day dosage level were found dead on study day three. The cause of one of the deaths was aspiration pneumonia. The cause of death of the other animals was not determined. The males in the 6000 and 3000 mg/kg/day dosage level groups exhibited a lower mean body weight gain and had lower body weights at termination (314 +/- 18.1 g and 331 +/-27.1 g, respectively) than controls (358 +/- 31.1 g). The mean body weights of the males of the 1000 mg/kg/day dosage level and of the treated female rats were comparable to controls throughout the study.
Pharmacotoxic signs noted for male and female animals in the 3000 and 6000 mg/kg/day dosage level groups included primarily yellow discoloration of fur in the anogenital region and soft stool. In addition, male animals in the 6000 mg/kg/day dosage level group exhibited yellow discoloration of fur - abdominal region and soft stool. One male animal in the 6000 mg/kg/day dosage level group was noted for hypoactivity, decreased grasping reflex, extremities hypothermic to touch, and ataxia on study day 3. Other signs noted among rats of various dosage level groups, or controls, were considered incidental and unrelated to the test article.
No toxicologically significant organ weight changes occurred in this study. Macroscopically, one male from the 6000 mg/kg/day dosage level group was found at the post-mortem examination to have multiple yellowish linear macroscopic lesions in the right testis. Microscopically, the lesions were found to be moderate bilateral mineralization of testes. Microscopically, treatment related mild mineralization in the heart was seen in this male and another male in the 6000 mg/kg/day dosage level group. Mineralization in the testes and heart were considered to be test article-related lesions. No other macroscopic or microscopic findings were considered to be related to the test article.

Key result

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: General toxicity

Key result

Critical effects observed:

no

Conclusions:

Effects on body weight of the 3000 mg/kg males were regarded as not adverse due to the relative low strength of effect (-7.5%) and the solitary appearance of this effect in this treatment.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer reviewed database.

Principles of method if other than guideline:

Performed after the NTP protocol

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: gavage

Details on oral exposure:

Ninety five male and 91 female B6C3F1 mice were acclimated for a period of 14 days before treatment. Based on daily observations and a preinitiation health verification conducted one week prior to study initiation, all animals were suitable for placement on the study (except 4 females whose water bottles had malfunctioned). A gross necropsy was performed on five males and 5 females one day prior to test initiation. All rats examined were found to be disease and parasite-free. Animals were randomly allocated to 4 groups of 10 animals/sex. They were 50-57 days old at study initiation. Males weighed 19.4 - 24.3 g and females weighed 16.0 - 20.8 g. Food and water were available ad libitum.
At study termination, serum samples from 5 mice/sex from the control groups were analyzed for the presence of antibodies to murine viruses designated by the NTP. Positive titers to both Sendai and PVM virus were detected in all mice using a hemagglutination inhibition assay.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative solutions were analyzed immediately after preparation at two labs. A sample taken from the 1000 mg/kg dosage level solution was analyzed to be 110% of the target concentration at one lab and 97% of the target concentration at the other lab.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

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

Remarks:

(referring to 100 % substance)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

(referring to 100 % substance)

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

Remarks:

(referring to 100 % substance)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: no

Sacrifice and pathology:

All survivors were euthanized on day 91 and received complete post-mortem examinations. The brain, lung, heart, thymus, liver, right testis and right kidney were weighed and relative weights (to brain and body) were calculated. These tissues, plus the pituitary, eyes, nasal cavity and turbinates, oral cavity, larynx and pharynx, tongue, salivary gland, Zymbal's gland, trachea, thyroid, parathyroid, mandibular lymph node, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, sternum (with marrow), aorta, bone marrow (femur), costochondral junction (rib), spinal cord, mammary gland, skin, gall bladder, mesenteric lymph node, pancreas, spleen, adrenal, urinary bladder, testes with epididymis, tunica of the testis and scrotal sac, seminal vesicles, prostate, ovary, uterus, thigh muscle, blood smear, sciatic nerve, gross lesions, tissue masses or suspect tumors and regional lymph nodes were saved in neutral 10% formalin. Stained sections of all collected organs from the high dose and control animals (except the larynx, tongue, Zymbal's gland, costochondral junction (rib), skin, seminal vesicles, thigh muscle and sciatic nerve) were examined histologically. The eyes and pharynx were only examined if grossly abnormal. If any lesion was thought to be related to treatment, the organ exhibiting this lesion was examined in the low and mid-dose animals.

Other examinations:

At study termination, serum samples from 5 mice/sex from the control groups were analyzed for the presence of antibodies to murine viruses designated by the NTP. Positive titers to both Sendai and PVM virus were detected in all mice using a hemagglutination inhibition assay.

Statistics:

Statistics: Body and organ weight data of treated animals were compared to controls using a one-way analysis of variance, Bartlett's test for homogeneity of variances and the appropriate t-test (for unequal and equal variances) as described by Steel and Torrie. Dunnett's multiple comparison tables were used to assess significant differences at p < 0.05.

Details on results:

Mortality: 1 male animal in 3000 mg/kg/day group died during the third week of treatment from a dosing accident, which initiated an abscess in the thoracic region.
Body weight gain: All dosed females showed an increased weight compared to the controls; the males of the 1000 and 6000 mg/kg/day group had the same or increased weight compared to the controls. The 6000 mg/kg/day group and controls showed no microscopically visible changes (the animals of the 1000 and 3000 mg/kg/day doses were not examined). Males and females in the control (2/10 and 4/10, respectively) and 1000 mg/kg groups (8/10 males and 8/10 females) exhibited alopecia.
Chronic interstitial pneumonia in the control and animals of the 6000 mg/kg/day group was seen in correlation with the positive finding of the Sendai-virus.

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest dose tested

Key result

Critical effects observed:

no

Conclusions:

Effects were regarded as not adverse and not related not the treatment, respectively.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 5 AUG 2010 to 24 APR 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Deviations:

not applicable

GLP compliance:

yes

Remarks:

according to Swiss Ordinance [SR 813.115.1]

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- strain: RccHan:Wistar
- Source: Harlan Laboratories, B.V., Horst, The Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: group means males: 291-319 g; group means females: 185-218 g
- Fasting period before study: no
- Housing:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
Afterwards animals were housed individually in Macrolon cages (Type III).
- Diet: pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, Kaiserazgst, Switzerland, batch no. 22/10), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-75.8
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(highly purified)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- formulations (w/v) were prepared weekly
- storage at room temperature (20 +/-5 °C)
- homogeneity of the test item in the vehicle was maintained during daily administration period using a magnetic stirrer

VEHICLE
- vehicle: phighly purified water
- dose volume: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical study was performed to check accuracy of preparation and to determine stability and homogeneity of test substance preparation (HPLC coupled to an ELSD detector).

Duration of treatment / exposure:

F0-males:
- at least 28 days (14 days pre-pairing, 14 days maximum during mating, until the day before sacrifice)
F0-females:
- approx. 7 weeks (2 weeks prior to mating, throughout mating (14 days maximum), and approx. 21 days gestation and at least up to day 4 post partum, and including the day before sacrifice)

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 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 results of a dose range finding study with the test item (in Han Wistar rats, Harlan Laboratories Study, using dose levels of 100, 300 and 1000 mg/kg/day).

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- clinical signs time schedule: daily cage-side observation (during acclimatisation up to necropsy), females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
-detailed clinical signs time schedule: Once prior to the first administration of the test item and weekly thereafter, observations were performed outside the home cage.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, but not during the mating period (in detail: Males: Weekly during pre-pairing and after pairing periods; Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.)

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day before or on day of scheduled necropsy (males), at day 5 post partum (lactating females)
- Anaesthetic used for blood collection: Yes (light isoflurane anaesthesia)
- Animals fasted: Yes (approx. 18 hours)
- How many animals: 5 males and 5 females from each group
- Parameters: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day before or on day of scheduled necropsy (males), at day 5 post partum (lactating females)
- Animals fasted: Yes (approx. 18 hours)
- How many animals: 5 males and 5 females from each group
- Parameters: alanine aminotransferase, aspartate aminotransferase, total bilirubin, total cholesterol, triglycerides, creatinine, glucose, urea, total protein, albumin, globulin, albumin globulin ratio, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once, males shortly before scheduled sacrifice and females during lactation on day 3 or day 4 post partum
- Dose groups that were examined: all dose groups
- Battery of functions tested: hearing ability / pupillary reflex / static righting reflex / grip strength / motor activity

OTHER:
Observations on females and litters, calculation of reproduction parameters are reported in section 7.8.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (with special attention to the reproductive organs)
HISTOPATHOLOGY: Yes
- from all males: prostate, testes, seminal vesicles with coagulating gland, epididymides
- from all females: ovaries
- from 5 animals/sex/group: any gross lesions, adrenal glands, brain, heart, kidneys, liver, spleen, thymus, spinal chord, small and large intestines,, stomach, thyroids, trachea and lungs (inflation and immersion), uterus with vagina, urinary bladder, lymph nodes (mandibular, mesenterical), peripheral nerv (sciatic), bone marrow

Other examinations:

ORGAN WEIGHTS
- from all males: testes, epididymides
- from 5 animals/sex/group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus

Statistics:

- means and standard deviaations were calculated
- Dunnett-test
- Steel-test
- exact Fisher-test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no effects on food consumption

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Parent Animals
CLINICAL SIGNS AND MORTALITY
All animals survived until the scheduled necropsy.
No clinical signs were observed during the whole study.
No findings were observed during the detailed weekly clinical observations of males and females.

BODY WEIGHT AND WEIGHT GAIN
Males:
Pre-pairing and Pairing Periods: Mean body weight and mean body weight gain were not affected by the treatment with the test item during the entire duration of the study. The lower statistical significances which occurred in group 2 on day 10 of the pre-pairing and on day 3 of the pairing period were considered to be incidental since there was no dose-dependency. In order of ascending dose level, overall body weight gain was 13%, 12%, 12% and 12% during the pre-pairing period and 4%, 5%, 5% and 4% during the pairing period.
Females:
Pre-pairing, Pairing, Gestation and Lactation Periods: No test item-related effects were observed in mean body weight and gains during the pre-pairing, pairing, gestation and lactation periods. The statistical significances noted during study were isolated occurrences or not following a dose-dependent pattern. In order of ascending dose levels, mean body weight gain was 9%, 10%, 9% and 7% during the pre-pairing, +61%, +58%, +61% and +59% during the gestation, and -5%, -4%, -5% and +2% during the lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males:
Pre-pairing Period: No statistically significant differences were observed in mean food consumption at any dose level.
During the pre-pairing period, mean food consumption compared to the control was -3.3%, -0.8% and -2.1% in groups 2, 3 and 4, respectively.
Females:
Pre-pairing, Gestation and Lactation Periods: No statistically significant differences were observed in mean food consumption during the prepairing,
gestation and lactation periods. In order of ascending dose levels, mean food consumption compared to the control was: +2.3%, -1.8% and +3.5% during the pre-pairing period, +0.5%, +2.8% and +4.6% during the gestation and +4.8%, -4.8% and +2.7% during the lactation period.

HAEMATOLOGY
Males
The assessment of the hematology data did not reveal any test item-related effects in males.
Females
The assessment of the hematology data did not reveal any test item-related effects in females. The statistically significant higher level of hematocrit (+5% compared to the control) in group 2 and the statistically significant lower partial thromboplastin time (-6.0% compared to the control) in group 3 were considered to be incidental since there was no dose-dependency.

CLINICAL CHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females.

NEUROBEHAVIOUR
None of the parameters under investigation during the functional observational battery gave indication of a test item-related effect.
In males, statistically significant higher body temperature in group 4 (38.8 °C compared to 38.4 °C in the control group) was within the range of the historical control data. In females, body temperature was similar in all groups.
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor and did not give any indication of a test item-related effect.

ORGAN WEIGHTS
No test item-related changes were observed in mean absolute and relative organ weight in males and females. In group 3 the statistically significantly higher relative organ weight (relative to body weight) of heart, liver and kidney were considered to be incidental since there was no dose dependency.

GROSS PATHOLOGY
Males
In group 4, two males (nos. 32 and 35) had uni- or bilateral pelvic dilation of kidneys, one male (no. 31) had spleen with a constriction and another male had nodular-thickened mesentery. In group 1, one male (no. 1) had pelvic dilation of kidneys, two males (nos. 6 and 8) had both testis and epididymidis reduced in size and one of these two males had also flaccid testis. The females mated with these males did not achieve a pregnancy.
Females
In group 4, dark red discoloration was observed in both ovaries of female no. 76 and in both uterine horns in female no. 80, and thickened cervix was noted in female no. 78. In group 3, female no. 61 had left ovary discolored dark red.
In group 2, dark red discoloration was observed in mandibular lymph node of female no. 53, in left ovary of female no. 54 and in both ovaries and uterine horns of female no. 59 and no. 60, this female had also a firm nodule in uterine adipose tissue. Cervix with watery cyst and dilation of
both uterine horns were observed in female no. 56.
In group 1, reddish discoloration of both ovaries and uterine horns were observed in female no. 46.

HISTOPATHOLOGY:
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
The histopathological evaluation of the reproductive organs did not reveal any test item-related changes in animals of the high-dose group, even in the evaluation with special emphasis on stages of spermatogenesis and histopathology of interstitial cell morphology. Higher degree of oligospermia in epididymides was recorded in male no. 6 and 8 in control group which was associated with tubular atrophy of the testes. Nothing special related to infertility was observed in prostate glands, seminal vesicles and coagulating glands of these infertile males.

OTHER:
There were no treatment related findings on reproduction. Details are presented in section 7.8.

Key result

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: see 'Remark'

Key result

Critical effects observed:

no

Conclusions:

There were no changes in clinical appearance, functional observations, locomotor activity, body weights, food consumption, clinical laboratory investigations, reproduction, litter observation, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Wistar rats (10/sex/dose) was performed according to OECD guideline 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day. Oral dosing of male and female Wistar rats with the test item for at least 28 days (males, females were treated up to approx. 7 weeks), revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development. From the results presented (absence of adverse effects) in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 000 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

Additional information

Male and female Fisher rats were subjected to test subchronic toxicity of DMDHEU (a surrogate substance to the submission substance). Doses of 1000, 3000, and 6000 mg/kg bw/day (10 males and 10 females /dose) were applied orally via gavage at 5 days/week for 13 weeks according to NTP-study protocol (NTP, 5701-307, 1983).

In the rats (NTP, 5701 -307, 1983), mean body weight gain was retarded in the male top and median dose group, but not in females. No significant differences were seen at the 1000 mg/kg bw/day dose level in both sexes. Yellow discoloration of the fur in the abdominal/ anogenital region and soft stool were prominent at the higher doses. One male in the high-dose group was noted for hypoactivity, decreased grasping reflex, hypothermic extremities, and ataxia. Other clinical signs at various dose levels were considered incidental and unrelated to the test article. No toxicologically significant organ weight changes occurred. At macroscopic post-mortem examination, no specific lesions were detected, but one high-dosed male having multiple yellowish linear macroscopic lesions in the right testis. Histopathological inspection revealed no specific treatment-related organ lesions, except that two males of the 6000 mg/kg dosage level group suffered from mild mineralization in the heart, and one from moderate bilateral mineralization of the testes, both phenomena considered substance-related. No such damage was seen in the 3000-mg groups.

In mice, mean body weight gain was equal or significantly higher than the control. Microscopic examination of the tissues from mice of the control and 6000 mg/kg groups gave no indication to treatment-related morphological changes . Chronic interstitial pneumonia was not influenced or induced by DMDHEU because in both the control and the high-dose group this disease was correlated with serum positivity of the Sendai virus (IRDC, 5701 -303, 1983).

Conclusions: Available experimental data are based on reliable studies conducted within the scope of the toxicological program under the auspices of U.S. NTP. The results clearly confirm the low toxicity already noted under single-dose conditions. The subchronic NOAEL for oral application was 3000 mg/kg in rat and 6000 mg/kg in mice (as 100% substance each).”

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the submission substance administered by oral gavage in Wistar rats (10/sex/dose) was performed according to OECD guideline 422. The dose levels for this study were 0, 100, 300 and 1000 mg/kg/day. Oral dosing of male and female Wistar rats with the test item for at least 28 days (males, females were treated up to approx. 7 weeks), revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development. From the results presented (absence of adverse effects) in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.

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 (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.