3,5-dimethylpyrazole

Endpoint summary

Description of key information

ORAL

Key 28 day study; Zmarowski (2012), performed to GLP, OECD 422 and EPA OPPT 870.3650, Klimisch 1, NOAEL = 20 mg/kg b.w./ day in the rat. target organ is the liver.

INHALATION

A data waiver has been provided to cover this endpoint.

DERMAL

A data waiver has been provided to cover this endpoint.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 2012 to 04 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed to valid guidelines and conducted under GLP conditions.

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 (Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Protocol Deviations:
1. The mean measured concentration of Group 2 samples was 111% of the target concentration and was outside the acceptance criteria for solutions (90-110%).
Evaluation: The deviation is only slightly outside the criterion. Since the variation coefficient was small, this was considered acceptable.
2. Temporary deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
3. On 02 and 21 August the functional observations and motor activity tests were performed before clinical observations and on 9 July and 10 August the clinical observations were performed outside the 1-3 hour window after dosing.
Evaluation: A difference in the observation order on these two days has no impact on the study. Furthermore, clinical signs would likely be observable outside of the 1-3 hour window, and thus there was no impact on the study’s integrity.
5 During the lactation period, no clinical observations were registered in the computer for pup 4 of litter 45 (Group 1) on Day 3 and the coagulating glands from male no. 35 were not available for histopathology.
Evaluation: Sufficient data is available for a thorough evaluation.
6. Animal nos. 63, 78 and 42 are necropsied after dosing on Day 27 (nos. 63 and 78) and Day 26 (no. 42) post-coitum.
Evaluation: These animals did not require fasting beforehand; this does not affect the study’s integrity.
7. Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 3.25 hours.
Evaluation: The fasting period was only slightly longer and was considered not to have adversely affected any findings. Current animal welfare approved practice allows a maximum fasting of 24 hours, which was not yet effective at the time of issuing the protocol.
8. Functional observational and motor activity data were collected for female no. 60 though they were not needed.
Evaluation: This just adds extra information and has no adverse impact on the study. These data are reported in Appendices 1 and 2.

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 11 weeks. Females were nulliparous and non-pregnant.
- Weight at study initiation: All animals were within ± 20% of the sex mean; males ranged from 287 to 327 g and females ranged from 187 to 218 g.
- Housing: Animals were housed in plastic cages, approximately 18 cm high. Cages contained sterilised sawdust as bedding material with paper as cage enrichment and nesting material.
> Pre-mating: Animals were housed by dose group in groups of 5 animals/sex/cage.
> During mating: Animals were housed in mating pairs, 1:1 by dose group.
> Post mating: Males were house in groups of 5/cage, females were housed individually.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least five days prior to treatment.
- Water, diet, bedding and cage enrichment was evaluated for contaminants and/or nutrients. There were no findings which could affect the results of the study.
- Health: The health of each individual was checked prior to study initiation, to ensure all animals were in good state health.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C.
- Humidity: 40 to 70%.
- Air changes: Approximately 15 room air charges per hour.
- Photoperiod: A 12 hour light/12 hour dark cycle was maintained.

IN-LIFE DATES: From: 09 July 2012 To: 03 September 2012.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Method: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustments were made for specific gravity of the vehicle (1.036). No corrections were made for the purity of the test material.
- Dose volume: 5 mL/kg b.w. based on the latest body weight meaurment.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was chosen based on trial formulations performed by the research laboratory.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Sampling: Test solutions were sampled and analysed once during the course of the study. All concentrations were sampled to confirm the accuracy of each preparation. Samples of the highest and lowest concentration were analysed for homogeneity and stability in the vehicle. Stability was confirmed at room temperature over a 6 hour period.
- Criteria: The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
- Results:
> Accuracy of preparation: The mean concentration of the 20 mg/kg b.w./day test solution was slightly outside the criterion, i.e. 111%. This was considered acceptable. The concentrations analysed in the 60 and 200 mg/kg b.w./day test solutions were in agreement with target concentrations, i.e. the mean accuracies were between 90% and 110%. No test substance was detected in the 0 mg/kg b.w./day formulation.
> Homogeneity: The 20 and 200 mg/kg b.w./day were homogeneous, i.e. the coefficient of variation was ≤ 10%.
> Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Total exposure ≥ 28 days.
Males: Exposed for 29-31 days, i.e. 2 weeks prior to mating, during mating and up to the day prior to scheduled necropsy.
Females: Exposed for 45-56 days, i.e. 2 weeks prior to mating, during mating, coitum, and at least 4 days of lactation (up to the day prior to scheduled necropsy). Two of the females were not dosed during littering.

Frequency of treatment:

Animals were dosed daily 7 days a week, at approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Remarks:

Doses / Concentrations:
20, 60 and 200 mg/kg b.w./day
Basis:
actual ingested

No. of animals per sex per dose:

Ten males and ten females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose concentrations were selected based on the findings of a 10 Day dose range finding study performed at 300 and 1000 mg/kg b.w.

Positive control:

None

Observations and examinations performed and frequency:

MORTALITY/VIABILITY OBSERVATIONS: Yes
- Time schedule: Recorded at least twice daily.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were made daily; detailed clinical observations were made 1-3 hours after dosing, including once prior to study initiation and at weekly intervals during the treatment period. Observations were performed outside of the cage in a standard arena.
- Scoring: The onset, grade and duration of each observation were recorded. Observations were graded 1 to 4 dependant on severity; 1 slight, 2 moderate, 3 severe, 4 very severe. Some clinical signs were scored dependant on their presence 1, or their absence 0.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were measured on the first day of exposure and then at weekly intervals thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and Day 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Measurements were recorded weekly, except during mating and for females without evidence of mating. Consumption for mated females were recorded on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Examinations were recorded daily starting from Day 12, except when animals were housed in pairs during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from selected animals between 0700 and 1030 hours in week 4 for males and at the end of lactation for females.
- Method of collection: Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, a maximum of 23.25 hours before sampling.
- How many animals: 5 animals/sex/group.
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the five selected animals between 0700 and 1030 hours in week 4 for males and at the end of lactation for females.
- Method of collection: Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared Li-heparin for clinical chemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
- Animals fasted: Yes
- How many animals: 5 animals/sex/group.
- Parameters checked in Table No. 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Five males and five females were selected from each exposure group and examined prior to necropsy, week 4 for males and towards the end of lactation for females. Examinations were performed after clinical observations and prior to blood sampling.
- Dose groups that were examined: All dose groups.
- Battery of functions tested: Hearing ability, papillary reflex, static righting reflex, grip strength, locomotor activity.
- Scoring: All examinations with the exception of locomotor were scored for their presence (normal) 0 or absence 1. Locomotor activity was scored according to the observed movements and ambulation.

Sacrifice and pathology:

SACRIFICE:
All males and the five selected females/group were subjected to necropsy. Animals were fasted for a maximum of 23.5 hours prior to necropsy then anaesthetised using isoflurane and subsequently exsanguinated.
Schedule;
> Females which delivered pups: Lactation Days 5-7.
> Females which failed to deliver pups with evidence of mating: Post-coitum Days 25-27.
> Females which failed to deliver pups without evidence of mating: Approximately 21 days after the last day of the mating period.
> Females with total litter loss: Within 24 hours of litter loss.
> Males: Following completion of the mating period, a minimum of 28 days of exposure.

GROSS PATHOLOGY: All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissue and organs, with special attention being made to the reproductive organs. Tissues were collected and fixed in 10% buffered formalin, see Table 2 descriptions on the tissues and organs examined.

ORGAN WEIGHTS: The following organs were examined organ weights and terminal body weight were recorded in five selected animals/sex/group; adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles (including coagulating glands) and the thyroid (including parathyroid). The epididymides and testes were examined in all remaining males.

HISTOPATHOLOGY: All organ and tissue samples were processed and embedded and cut at a thickness of 2-4 mm and then stained with haematoxylin and eosin, see Table 3 for details of examinations. The histopathology data was peer reviewed by a second pathologist.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant findings.

Mortality:

no mortality observed

Description (incidence):

No toxicologically relevant findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant reduction observed at 200 mg/kg.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduction was observed at 200 mg/kg.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

An increase in water consumption was observed at concentations ≥ 60 mg/kg.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes at 200 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Toxicologically statistically significant changes at 200 mg/kg.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights were affected at 200 mg/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effects observed at 200 mg/kg.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effects were observed at 60 and 200 mg/kg.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: No treatment related mortalities occurred during the course of the study. One female was euthanized in the highest concentration group, 200 mg/kg, this was due to total litter loss.
- Clinical signs: Piloerection was noted for three females from the 200 mg/kg exposure group and one female at 20 mg/kg, however this was only noted for a limited number of days in each animal and the reaction was slight.
Incidental findings include alopecia, salivation (60 mg/kg) and pale appearance (200 mg/kg). These effects occurred in low incidences and thus were not considered to be toxicologically relevant.
No clinical signs were reported for males in either the control group or any of the treatment groups.
Females from the control group displayed alopecia, this was first observed during the preproduction period on Day 1 of Week 3. The reaction lasted until Day 2 of Week 5 and was assigned a grade of 1, slight. The reaction was observed in 5 to 15% of the animals, which rose to between 15% and 25% on the last day (Day 2 of Week 5).
Piloerection was observed for females in the 20 mg/kg exposure group, the reaction was observed in the reproduction period on Day 4 of Week 2. The reaction was recorded for one day and was assigned a grade of 1, occurring in between 5% and 15% of the animals.
Salivation was observed in the females of the 60 mg/kg treatment group during premating on Day 1 of Week 1. Salivation was assigned a grade of 1 and observed in 5 to 15% of the animals.
Females from the 200 mg/kg group were first observed for piloerection on Day 2 of Week 2 lasting until Day 5 of week 4, the median value for the highest individual reaction never exceeded 1, slight. The reaction was observed in between 5 and 15% of the animals with the exception of Day 4 and 7 of Week 2 where the reaction was observed in between 15 and 25% of the animals. A pale appearance was observed in between 5 and 15% of the animals on Day 3 of Week 4.

BODY WEIGHT AND WEIGHT GAIN
- Body weights and body weight gains were significantly lower for males at 200 mg/kg than controls on Day 8 of the premating and through the entire mating period. Body weight gain was significantly lower for females on Day 8 of the premating period and on Day 11 of the post coitum period. Body weight gain was also slightly (not significantly) lower on Days 7, 14-17 and 20 of the post coitum period compared to controls.

FOOD CONSUMPTION
- Absolute food consumption was lower for males at 200 mg/kg from Days 1-8 of the premating period while relative food consumption was lower during the entire premating period. For females, relative food consumption was slightly lower during Days 1-8 of the premating period, and Days 17-20 of the post coitum period. Absolute and relative food consumption were both significantly lower than controls from lactation Days 1-4.

WATER CONSUMPTION
- At 200 mg/kg water consumption was increased for males during the entire treatment period (with the exception of Days 3-5 of the mating period) while water consumption was increased for females during the entire premating and post coitum periods (not always statistically significant).
Water consumption was also higher for females at 60 mg/kg over several days during the post coitum period, though the difference from controls was never statistically significant, a relationship to treatment could not be excluded.

HAEMATOLOGY
At 200 mg/kg, the following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
-Decreased red blood cells (RBC; both sexes)
-Decreased haemoglobin (both sexes, also for males at 60 mg/kg)
-Increased mean corpuscular volume (MCV; both sexes)
-Increased mean corpuscular haemoglobin (MCH; both sexes)
-Decreased mean corpuscular haemoglobin concentration (MCHC; both sexes)
-Increased prothrombin time (PT; both sexes)
-Decreased haematocrit (females)
-Increased reticulocytes (males)
-Increased red blood cell distribution width (RDW; males)
-Increased activated partial thromboplastin time (APTT; males, not statistically significant)

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished animals treated at 200 mg/kg from controls:
-Increased alanine aminotransferase (ALAT; males)
-Increased total bilirubin (both sexes)
-Increased bile acids (both sexes)
-Decreased sodium (males)
-Increased potassium (females)
The significant increase in creatinine and the significant decrease in glucose seen for females at 20 and 60 mg/kg, respectively, were not considered toxicologically relevant as they occurred in the absence of a treatment-related distribution.

NEUROBEHAVIOUR
- The pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
- Hearing ability: At 200 mg/kg none of the animals tested, both male and female, showed a response to the hearing ability test. The observation was shown to be significant at 5% using the steel-test for significance. While the animals could hear, they did not react to the auditory stimulus used during this test. Thus, this deficit represented more of a failure to react to the stimulus, and not an inability to hear. A reaction was not observed in any of the other treatment groups.
- Locomotor activity: The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
- At 200 mg/kg lower terminal body weights, testes (absolute and relative), seminal vesicles (absolute and relative), prostate (absolute), epididymides (absolute) and increased spleen (absolute and relative), thyroids (relative) and kidney (relative) weights were noted for males while lower thymus (absolute and relative) and higher spleen (absolute and relative) weights were noted for females.
- Absolute and relative spleen weights were significantly increased for animals at 20 and 60 mg/kg as well. As no corresponding adverse effects in the spleen were noted for these groups during the microscopic examination, these increased weights were not considered toxicologically relevant.
- The reduced absolute brain weights seen for females at 200 mg/kg were considered secondary to their slightly lower body weights since their brain to body weight ratios were not significantly different than controls.
- The significant increase in seminal vesicle weight seen for males at 60 mg/kg was not considered toxicologically relevant as it occurred in the absence of a dose response (these weights were reduced for males at 200 mg/kg) and remained within the range considered normal for this age and strain.

GROSS PATHOLOGY
- At 200 mg/kg an enlarged spleen and reduced size of the thymus was seen for 6/10 and 5/10 females. Reduced size of the epididymides, testes and seminal vesicles was noted for single males. Despite the limited incidence, microscopic examination confirmed that these were treatment related.
- Incidental findings seen for animals of the control and treated groups included uterus contains fluid, pelvic dilation of the kidney, alopecia on the foreleg, tan focus or discoloration on the left clitoral gland, yellow or soft nodule on the head of the epididymides. These observations were within the background range of findings that are encountered among rats of this age and strain. As they did not show a dose related incidence trend, they were not considered to be toxicologically relevant.

HISTOPATHOLOGY
Treatment-related microscopic findings were seen for animals at 60 and 200 mg/kg, and consisted of:
- Thymus: Increased severity of lymphoid atrophy in males (3/5, minimal) and females (5/5, up to moderate) treated at 200 mg/kg.
- Liver: Hepatocellular basophilia (up to slight) and/or apoptosis/single cell necrosis (up to marked) in the area directly around the central veins in males treated at 60 mg/kg and in both sexes treated at 200 mg/kg. Additionally, males treated at 200 mg/kg showed hepatocellular karyomegaly (5/5, up to slight) in the same area and midzonal hepatocellular vacuolation (3/5, up to slight).
- Spleen: An increase in severity of hematopoietic foci in males (6/6, up to marked) treated at 200 mg/kg.
- Epididymides: Oligospermia (1/6, marked) and an increased incidence and/or severity of seminiferous cell debris (3/6, up to slight) treated at 200 mg/kg.
- Testes: Degeneration/depletion of spermatocytes (6/6, up to massive) and an increase in incidence and/or severity of spermatidic giant cells (5/6, up to moderate) treated at 200 mg/kg.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight, food and water consumption, haematology, clinical chemistry, organ weights, gross pathology and histopathology.

Critical effects observed:

not specified

Table 4a. Male Body Weights (g)

MALES		Dose Group (mg/kg)
		0		20		60		200
PREMATING		Body Weight	Weight Gain	Body Weight	Weight Gain	Body Weight	Weight Gain	Body Weight	Weight Gain
Day 1, Week 1	Mean (SD)	313 (6.4)	0 (0.0)	312 (10.9)	0 (0.0)	311 (8.6)	0 (0.0)	315 (7.4)	0 (0.0)
Day 8, Week 2	Mean (SD)	340 (8.4)	9 (1.2)	344 (11.3)	10 (1.8)	338 (13.3)	9 (2.0)	326 (10.5)*	3 (3.4)**
MATING PERIOD
Day 1, Week 1	Mean (SD)	365 (9.5)	17 (1.6)	373 (12.6)	19 (2.2)	366 (15.4)	18 (2.5)	344 (12.6)**	9 (3.7)**
Day 8, Week 2	Mean (SD)	380 (8.7)	21 (1.6)	387 (14.5)	24 (2.6)	380 (19.1)	22 (3.5)	356 (12.9)**	13 (4.0)**
Day 15, Week 3	Mean (SD)	400 (10.0)	28 (1.9)	408 (16.6)	31 (3.6)	401 (21.3)	29 (4.2)	370 (13.4)**	17 (4.5)**

10 Males were weighed in each group.

* Significant at 5%

** Significant at 1%

Table 5. Female Body Weight (g) and Weight Gain (%)

FEMALES		Dose Group (mg/kg)
		0		20		60		200
PREMATING		Body Weight	Weight Gain	Body Weight	Weight Gain	Body Weight	Weight Gain	Body Weight	Weight Gain
Day 1, Week 1	Mean (SD)	203 (6.3)	0 (0.0)	203 (7.4)	0 (0.0)	201 (6.7)	0 (0.0)	206 (9.2)	0 (0.0)
	No.	10	10	10	10	10	10	10	10
Day 8, Week 2	Mean (SD)	206 (8.1)	2 (3.3)	206 (9.1)	1 (2.5)	207 (8.2)	3 (2.8)	204 (10.1)	-1 (2.1)*
	No.	10	10	10	10	10	10	10	10
MATING PERIOD
Day 1, Week 1	Mean (SD)	216 (11.0)	7 (5.1)	218 (9.2)	7 (3.3)	218 (6.8)	9 (3.7)	218 (11.3)	6 (3.5)
	No.	10	10	10	10	10	10	10	10
Day 8, Week 2	Mean (SD)					239	20	220 (14.5)	10 (1.3)
	No.					1	1	3	3
Day 15, Week 3	Mean (SD)							217 (12.0)	11 (0.3)
	No.							2	2
Day 22, Week 4	Mean (SD)							227	12
	No.							1	1
Day 29, Week 5	Mean (SD)							235	16
	No.							1	1
Day 36, Week 6	Mean (SD)							230	13
	No.							1	1
POST COITUM
Day 0	Mean (SD)	217 (9.1)	0 (0.0)	216 (11.2)	0 (0.0)	225 (10.4)	0 (0.0)	222 (10.8)	0 (0.0)
	No.	8	8	9	9	9	9	6	6
Day 4	Mean (SD)	230 (11.3)	6 (1.3)	230 (13.8)	7 (2.2)	238 (10.9)	6 (2.5)	232 (12.9)	5 (2.5)
	No.	8	8	9	9	9	9	6	6
Day 7	Mean (SD)	238 (10.5)	10 (1.4)	238 (14.7)	10 (2.8)	247 (13.3)	10 (4.4)	233 (15.2)	5 (5.3)
	No.	8	8	9	9	9	9	6	6
Day 11	Mean (SD)	255 (14.3)	18 (3.1)	259 (15.9)	20 (2.5)	265 (14.7)	18 (5.3)	248 (9.9)	12 (2.4)*
	No.	8	8	9	9	9	9	6	6
Day 14	Mean (SD)	264 (14.9)	22 (3.9)	269 (18.3)	24 (3.3)	277 (17.2)	24 (6.2)	261 (13.9)	18 (4.2)
	No.	8	8	9	9	9	9	6	6
Day 17	Mean (SD)	288 (17.3)	33 (5.7)	296 (23.1)	37 (4.9)	303 (18.3)	35 (6.7)	286 (9.5)	29 (5.1)
	No.	8	8	9	9	9	9	6	6
Day 20	Mean (SD)	326 (21.6)	50 (7.8)	333 (27.6)	54 (6.2)	343 (21.0)	53 (7.3)	317 (13.9)	43 (7.1)
	No.	8	8	9	9	9	9	6	6
LACTATION
Day 1	Mean (SD)	249 (17.1)	0 (0.0)	249 (15.2)	0 (0.0)	258 (13.1)	0 (0.0)	241 (13.2)	0 (0.0)
	No.	8	8	9	9	9	9	6	6
Day 4	Mean (SD)	260 (17.8)	4 (3.1)	258 (16.4)	3 (3.4)	269 (17.8)	4 (3.0)	247 (14.5)	4 (1.9)
	No.	8	8	9	9	9	9	5	5

Table 6. Relative Food Consumption (g/kg b.w./day)

Observation Period	Dose Group (mg/kg)
	0	20	60	200
Males
Premating period	66	69	66	59
Mating period	59	59	61	56
Females
Premating period	71	71	73	67
Post Coitum	76	74	79	75
Lactation	108	97	98	83

Values are the mean of the mean from each interval recorded.

 

Table 7. Mean Water Consumption (g/animal/day)

Observation Period	Dose Group (mg/kg)
	0	20	60	200
Males
Premating period	33	32	34	41
Mating period	31	32	33	37
Females
Premating period	24	23	26	34
Post Coitum	23	30	36	48
Lactation	44	40	46	44

Values are the mean of the mean from each interval recorded.

Conclusions:

Under the conditions of the test treatment related effects on body weights, food and water consumption, functional observations, clinical pathology, macroscopic findings and microscopic findings in the thymus, liver spleen, testes and epididymides were observed at 200 mg/kg/day. Females exposed at 60 mg/kg/day had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings at the microscopic examination. Based on these finding the NOEAL was considered to be 20 mg/kg/day.

Executive summary:

The repeat dose toxicity of the test material was determined in a 28 day oral study performed in line with GLP and the standardised guidelines OECD 422 and EPA OPPTS 870.3650.

Both male and female wistar rats were exposed to the test material at 0 (control), 20, 60 and 200 mg/kg b.w./day administered via oral gavage for ≥ 28 days. Animals were evaluated for mortality / viability, clinical signs (daily), functional observations and locomotor activity (males: Week 4, females: end of lactation), body weight and food consumption (at least at weekly intervals), water consumption (daily from Day 12 onwards, except when animals were paired for mating), clinical pathology (males: Week 4, females: end of lactation), macroscopy at termination, organ weights and histopathology on a selection of tissues. Test solutions were analysed once during the course of the study, where the accuracy of the preparations, homogeneity and stability were confirmed.

Under the conditions of the test, toxicity was observed at 60 and 200 mg/kg b.w./day. Treatment related effects on body weights, food and water consumption, functional observations, clinical pathology, macroscopic findings and microscopic findings in the thymus, liver spleen, testes and epididymides were observed at 200 mg/kg b.w./day. Females exposed at 60 mg/kg b.w./day had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings at the microscopic examination. There was no mortality observed up to the highest concentration tested during the study. Based on these findings the NOAEL was considered to be 20 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is performed in line with standardised guideline and is sufficient to justify a Klimisch score of 1, thus the dataset is considered to be of high quality.

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

Exposure via the oral route is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation and the dermal route are considered less appropriate and has been omitted on this basis. Additionally a sub-chronic toxicity study (90 days) does not need to be conducted as a reliable short-term toxicity study (28 days) is available. The short-term toxicity study Zmarowski (2012) gives a suitable oral NOAEL, allowing extrapolation to the 90 day NOAEL to address sub-chronic toxicity for the same route of exposure.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The key study (Zmarowski, 2012) was performed in compliance with GLP and the standardised guidelines OECD 422 and EPA OPPTS 870.3650. The study was performed to a good standard with a sufficient level of detail to assess the quality of the data presented. The study was assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Exposure via the oral route is an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation is considered less appropriate and has been omitted on this basis.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Exposure via the oral route is an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation is considered less appropriate and has been omitted on this basis.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Exposure via the oral route is an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via the dermal route is considered less appropriate and has been omitted on this basis.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Exposure via the oral route is an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via the dermal route is considered less appropriate and has been omitted on this basis.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The key study submitted to evaluate the repeated dose toxicity of the test substance was administered via the oral route. This is considered to be the most relevant route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. The study indicated that the test material requires classification in accordance with Regulation EC 1272/2008. The test material meets the criteria for classification as a STOT Repeated Exposure category 2, (H373: May cause damage to organs through prolonged or repeated exposure) with the liver as the target organ in accordance with Regulation (EC) No. 1272/2008.