Ethyl 2-methyl-1,3-dioxolane-2-acetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 February 2013 to 6 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl CD®(SD) IGS BR
- Age at study initiation: 10 weeks old (males); 9 weeks old (females)
- Weight at study initiation: 323 - 409 g (males); 204 - 277 g (females)
- Housing: Individually housed (except during mating) in polycarbonate cages with stainless steel lids and containing autoclaved sawdust.
- Diet: ad libitum
- Water: Filtered tap water (0.22 µm filter), ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

drinking water treated by reverse osmosis

Details on exposure:

PREPARATION AND ADMINISTRATION OF DOSING SOLUTIONS
Test material was formulated in vehicle at the required concentrations. The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dose volume of 10 mL/kg/day was used.
Control animals (group 1) received vehicle only.

Details on mating procedure:

Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night. Sibling pairings were avoided.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated day 0 post coitum.
Each female was placed with the same male until mating occurred or 14 days had elapsed.
Females were allowed to litter normally and rear their progeny until day 5 post partum.
The morning when the parturition was completed was designated day 1 post partum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in the dose formulations was analysed by GC-FID.

- Instrumentation: Varian Gas Chromatography system with FID detector
- Chromatographic conditions:
Column: HP-ULTRA (Agilent) 50 m x 0.32 mm (0.52 µm)
Mobile phase: hydrogen
Sensitivity: 12
Column temperature: from 70°C to 300°C at 10°C/min
Constant flow rate: 2 mL/min
Split rate: 10
Software: Empower 2 (Waters)
Detector temperature: 300°C
Injector temperature: 250°C
Injected volume: 2 µL
Detector gas flows: 30 mL/min for make up; 30 mL/min for hydrogen and 300 µL/min for air
Retention time: 8.4 min (test material); 8.8 min (internal standard)

Standard solutions of test material were prepared. An internal standard (1,4 diisopropylbenzene) was used to correct the injection variability.

- Preparation of Standard Solutions
Stock solutions of test material in diluent were prepared at 0.2, 0.02 and 0.0015 mg/mL.

- Preparation of Internal Standard
A solution of 1,4-diisopropylbenzene in methanol was prepared at 10 µg/mL.

ANALYTICAL METHOD
1 mL of dose formulation was diluted with diluent to reach the nominal concentration for injection. The diluted samples were analysed by GC-FID, bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions. The internal standard was used to correct the injection variability.

ANALYSIS OF DOSE FORMULATIONS
The test material concentrations in the administered dose formulations analysed in weeks 1, 4 and 6 remained within an acceptable range of -4.4 to +8.69 % when compared to the nominal values. Test material was not detected in the control samples.

Duration of treatment / exposure:

Males received dose formulations for 2 weeks before pairing, during the 2 week pairing period, until sacrifice (at least 5 weeks in total). Females received dose formulations for 2 weeks before mating, during the 2 week pairing period, during gestation, and during lactation until day 5 post partum inclusive (or until sacrifice).

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the selected dose levels were based on results of previous studies 2 acute oral studies where the LD50 values were > 5000 mg/kg and a 2-week preliminary toxicity study, by the oral route, in rats in which no animals died at dose levels up to 1000 mg/kg bw/day, however, some clinical signs were noted.
- Rationale for animal assignment: during the acclimation period, the required number of animals was selected according to body weight and/or clinical condition. The animals were allocated to groups (by sex) according to a computerised stratification procedure based on body weight, so that the average body weight of each group was similar.

Therefore, 1000 mg/kg/day was selected as the high dose-level for this study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and morbidity was checked once daily before the treatment period and at least twice a day during the treatment period. General clinical observations were performed once a day after treatment at approximately the same time.

MATING
The pre-coital time was calculated for each female.

PARTURITION
Any sign of a difficult or prolonged parturition was recorded, and the length of gestation was calculated.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and weekly thereafter, observations were performed on all animals outside the home cage in a standard arena.
- Observations included (but were not limited to): changes in fur, skin, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the first day of treatment and weekly thereafter until sacrifice. Females were weighed on the first day of treatment then weekly until mated (or until sacrifice) and on days 0, 7, 14 and 20 post coital and days 1 and 5 post partum.
Bodyweight gain was calculated for selected intervals and for the overall study.

FOOD CONSUMPTION: Yes
- Food consumption by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period. The measures coincided with body weight measurements.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 post partum.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper at the next filling.

WATER CONSUMPTION: No

OTHER: haematology parameters, blood biochemistry parameters and neurobehaviour were also tested.

Oestrous cyclicity (parental animals):

The estrous stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females were mated.

Sperm parameters (parental animals):

- Seminology: Before sacrifice at the end of the pairing period, each male was anesthetised by an intraperitoneal injection of sodium pentobarbital. Analyses were performed on all groups.

- Epididymal sperm
The left epididymis of each male was removed, weighed and sperm from the cauda was sampled for motility and morphology investigations.
The cauda of the left epididymis were separated from the corpus using a scalpel and subsequently kept at -20°C pending further investigation

- Sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatoza from a sample of 200 spermatoza was evaluated under a microscope using a 40-fold magnification. Results expressed as the proportion of motile and non-motile spermatozoa.

- Sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatoza per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
> normal,
> normally shaped head separated from flagellum,
> misshapen head separated from flagellum,
> misshapen head with normal flagellum,
> misshapen head with abnormal flagellum,
> degenerative flagellar defect(s) with normal head,
> other flagellar defect(s) with normal head.

- Sperm count
After thawing, the left cauda epididymis was weighed and homogenised in a saline-triton solution using a Polytron. An aliquot of the suspension was samples and the number of spermatoza was counted in a microscope slide counting chamber. Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

- Testicular sperm head count
The left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell.
Results were expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).

Litter observations:

Each live up was identified individually on day 1 post partum.

LITTER SIZE
The total litter size and numbers of each sex were recorded as soon as possible after birth. Any gross malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalised pups.

CLINICAL SIGNS
The pups were observed daily for clinical signs or abnormal behaviour.

BODY WEIGHT
The body weight of each pup was recorded on days 1 and 5 post partum.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
A complete macroscopic examination was performed. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities/muscles with their associated organs and tissues and the neck with its associated organs and tissues. All gross lesions were recorded and preserved.
The number of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 post partum.

ORGAN WEIGHTS: Yes
The body weight of each animal sacrificed as scheduled was recorded before sacrifice.The wet weights of the following were recorded as soon as possible after dissection: adrenals, brain (including medulla/pons cerebellar and cereebral cortex), epidiymides, heart, kidneys, liver, ovaries (with oviducts), prostate, seminal vesicles, spleen, testes, thymus, thyroids with parathyroids and uterus (horns and cervix).
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY: Yes
All tissues listed below were from the first five sacrificed as scheduled males and the first five females which delivered and were sacrificed on day 6 post partum of the control and high groups (groups 1 and 4) and for the female sacrificed prematurely:
adrenals, brain, cecum, colon, duodenum, epididymides, esophagus, gut-associated lymphoid tissue, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, ovaries, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord, spleen, sternum with marrow bone, stomach with forestomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus and vagina.
Microscopic examination was also performed on all lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

Pups found dead and pups sacrificed on day 5 post partum were carefully examined externally for gross external abnormalities. No macroscopic examination was performed and no tissues were preserved.

Statistics:

Data was compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions). PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Reproductive indices:

- pre-implantation loss
[(no. of corpora lutea - no. of implantation sites) / no. of corpora lutea] x 100

- post-implantation loss
[(no. of implantation sites - no. of live pups) / no. of implantation sites] x 100

- mating index
[no. of mated animals / no. of paired animals] x 100

- fertility index
[no. of pregnant female partners / no. of mated pairs] x 100

- gestation index
[no. of females with live born pups / no. of pregnant females] x 100

- live birth index
[no. of live ups born / no. of delivered pups] x 100

Offspring viability indices:

- viability index on day 4 post partum
[no. of surviving pups on day 4 post partum / no. of live born pups] x 100

- lactation index in day 5 post partum
[no. of surviving pups on day 5 post partum / no. of surviving pups on day 4 post partum] x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths that could be attributed to treatment during the study.
In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions (piloerection, round back, swollen on left forelimb and chromorhinorrhea). At necropsy, a pouch was observed in the subcutaneous tissue of the left axillary region with a brown thick content which correlated histologically with acute inflammation. Inflammation with hemorrhage was seen in the larynx and particularly in surrounding tissues with edema and hemorrhage. There was no histological correlation with the perforation of the larynx noted at necropsy. Inflammation in tissues adjacent to the thymus was also observed. Additional changes consisted of a moderate hemorrhage in the left adrenal which correlated with a red discoloration at necropsy. Moderate increased apoptotic cell numbers was seen in the thymus along with decreased periarteriolar lymphoid sheaths (PALS) in the spleen. These observations in the lymphoid tissue were considered to be non specific, probably stress related. All these changes were suggestive of a gavage trauma due to the technical procedures (gavage error).
Ptyalism (hypersalivation) was recorded in males receiving 300 mg/kg/day (2/10 males) and in most males and females dosed at 1000 mg/kg/day during the premating period (9/10 males and 5/10 females) and in all females during the gestation and lactation period. This finding was considered to be related to the test material and a sign of discomfort but not an adverse effect.
A treatment-related effect was considered to be unlikely for the few other clinical signs as they are commonly observed in this species and strain or observed with no dose-level relationship.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no treatment-related effects in mean body weights and mean body weight changes. There were no biologically significant effects on mean food consumption associated with treatment with the test material.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no treatment-related effects on the estrous cycle of parental animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no treatment-related effects on epididymal sperm parameters (motility, morphology and count) and testicular sperm production rate (testicular sperm head count).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
With the exception of a single female dosed at 300 mg/kg/day, all animals mated during the first week of the pairing period. There was no effect on the mean number of days taken to mate and all females were pregnant.
There were no treatment-related effects on mating and fertility data. Furthermore, there were no treatment-related effects on delivery and litter data.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no changes in the mean organ weights which were suggestive of a test material-related effect. The small differences observed between groups were considered to reflect normal variations and were not considered to be due to treatment with the test material.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no changes at necropsy which were indicative of a test material-related effect. Any changes observed were considered to be part of the normal background commonly seen in rats.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no changes at histopathological examination which were suggestive of a test material-related effect.
In the mandibular lymph nodes, presence of sinusal red blood cells was seen at a higher incidence in females given 1000 mg/kg/day than in controls. As this observation is commonly seen in rats, was observed unilaterally in 3/5 treated females and in the absence of a similar trend in males, any relationship with the test material was excluded.
Tubular basophilia was seen in the kidneys of 2/5 females given 1000 mg/kg/day but not in controls. As this observation was focal and unilateral, in the absence of a similar trend in males, any relationship with the test material was excluded.
Other changes observed were considered to be part of the normal background commonly seen in rats and without relationship with the test material.
Careful examination of testes, epididymides and ovaries did not reveal any treatment related changes.

OTHER FINDINGS (PARENTAL ANIMALS)
There were no treatment related effects on functional observation battery observations or on the heamatology and blood chemistry parameters investigated.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were noted.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance - mating and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were noted.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

(mortality) see "details on results" for information

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "details on results" for information

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

MORTALITY (OFFSPRING)
At 1000 mg/kg/day, there was a tendency towards an increased number of pups found dead (2.6% vs. 0.7% in controls) and cannibalised pups (3.2% vs 1.4% in controls). However, when compared with Historical Control Data (2009-2012), it is unclear whether this was a test material-related effect.

CLINICAL SIGNS (OFFSPRING)
There were no test material-related findings at pup examination.

VIABILITY (OFFSPRING)
There were no toxicologically significant effects on live birth, viability and lactation indexes associated with treatment with the test material.

BODY WEIGHT (OFFSPRING)
At 1000 mg/kg/day, there was a tendency towards a decrease in mean pup body weight and mean body weight changes. This finding was considered to be test material-related but of minor toxicological significance as the difference was of low magnitude and remained within the range of 2009-2012 Historical Control Data.

OTHER FINDINGS (OFFSPRING): SEX RATIO
There were no treatment-related effects on sex-ratios (% of male pups). Furthermore, no macroscopically malformed pups were observed.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Table 1: Mating and Fertility Data

Dose level (mg/kg bw/day)	0	100	300	1000
No. of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
No. of males mated	10	10	10	10
No. of females mated	10	10(a)	10	10
Mean no. of days taken to mate	3.0	3.1	4.4	2.8
No. of pregnant females	10	9(a)	10	10
Male fertility index	100%	100%	100%	100%
Female fertility index	100%	90%(a)	100%	100%

(a) one female was prematurely sacrificed the day of its positive mating

M = male; F = female

Table 2: Delivery and Litter Data

Dose level (mg/kg bw/day)	0	100	300	1000
No. of pregnant females	10	9	10	10
No. of females which delivered	10	9	10	10
Mean duration of gestation (days)	21.3	21.3	21.6	21.5
Mean no. of corpora lutea	16.4	15.1	16.4	16.9
Mean no. of implantations	16.4	15.1	16.4	16.9
Mean pre-implantation loss (%)	0.0	0.0	0.0	0.0
Mean no. of pups delivered	14.5	14.2	15.0	15.4
Mean post-implantation loss (%)*	11.0	5.7	8.0	9.0
Viability index on day 4 post partum (%)	97.9	97.7	97.3	94.2

* manually calculated (no statistics performed)

Table 3: Pup Mortality Data

Dose level (mg/kg bw/day)	0	100	300	1000
No. of pups found dead	1	3	3	4
No. of pups cannibalised	2	2	1	5
No. of pups delivered	145	128	150	154

Table 4: Pup Body Weights (g)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Day 1 p.p.	7.0	7.1	7.1	6.7	6.5	6.5	6.7	6.4
Day 5 p.p.	11.3	11.7	11.4	10.7	10.9	11.1	11.0	10.3
Body weight change (g)	+4.4	+4.6	+4.3	+4.0	+4.3	+4.6	+4.2	+4.0

p.p. = post partum

Table 5: Summary of Epididymal Sperm Count and Motility

Dose Level (mg/kg/day)			0	100	300	1000
Number of Spermatozoa (10⁶/cauda of epididymis)		Mean	113.5	123.7	110.2	119.8
		SD	28.0	28.9	15.6	19.7
		N	10	10	10	10
Number of Spermatozoa (10⁶/g cauda of epididymis)		Mean	353.5	376.3	364.1	357.2
		SD	69.3	64.4	52.5	62.8
		N	10	10	10	10
Epididymal sperm motility (%)	Motile	Mean	94.1	95.9	89.9	90.5
		SD	7.4	3.1	8.7	10.8
		N	10	10	10	10
	Non-motile	Mean	5.9	4.2	10.1	9.6
		SD	7.4	3.1	8.7	10.8
		N	10	10	10	10

 

Table 6: Summary of Testicular Sperm head Count and Daily Sperm Production

Dose Level (mg/kg/day)		0	100	300	1000
Number of sperm heads (10⁶/g of testis)	Mean	111.4	117.9	110.1	112.7
	SD	11.9	16.1	10.3	16.0
	N	10	10	10	10
Daily sperm production rate (10⁶/g of testis/day)	Mean	18.3	19.3	18.1	18.5
	SD	2.0	2.6	1.7	2.6
	N	10	10	10	10

 

Table 7: Summary of Epididymal Sperm Morphology

Dose Level (mg/kg/day)		0	100	300	1000
n		10	10	10	10
Normal	Mean %	95.6	95.2	94.9	95.6
	SD	3.0	3.8	3.2	4.2
Normally shaped head separated from flagellum	Mean %	3.1	3.4	3.2	3.3
	SD	2.3	2.6	2.0	2.9
Abnormal head separated from flagellum	Mean %	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
Abnormal head with normal flagellum	Mean %	0.3	0.4	0.1	0.2
	SD	0.7	0.8	0.3	0.4
Abnormal head with abnormal flagellum	Mean %	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
Normally shaped head with abnormal flagellum	Mean %	1.0	1.0	1.8	0.9
	SD	1.2	1.1	1.5	1.1

 

Table 8: Summary of Male Body/Organ Weights

Organ		Dose Group (mg/kg/day) n = 10
		0	100	300	1000
Final Body Weight (n = 10)	Mean Weight (g)	417.3	432.0	447.1	443.8
Adrenal Glands (n = 5)	Mean Weight (g)	0.07580	0.07700	0.07740	0.08400
	Mean % Body	0.01766	0.01708	0.01768	0.01799
Brain (n = 5)	Mean Weight (g)	2.09	20.3	2.08	2.09
	Mean % Body	0.48609	0.45047	0.47657	0.44759
Epididymides (n = 10)	Mean Weight (g)	1.44	1.40	1.41	1.46
	Mean % Body	0.34573	0.32473	0.31715	0.33084
Heart (n = 5)	Mean Weight (g)	134	1.39	1.36	1.45
	Mean % Body	0.31160	0.30831	0.31112	0.31067
Kidneys (n = 5)	Mean Weight (g)	3.03	3.13	3.21	3.34
	Mean % Body	0.70657	0.69605	0.73149	0.71510
Liver (n = 5)	Mean Weight (g)	10.94	11.48	10.82	12.04
	Mean % Body	2.53	2.55	2.46	2.58
Prostate (n = 5)	Mean Weight (g)	1.52	1.31	1.33	1.58
	Mean % Body	0.34868	0.28918	0.30124	0.33704
Seminal Vesicles (n = 5)	Mean Weight (g)	1.66	1.84	1.84	1.77
	Mean % Body	0.38802	0.40978	0.42236	0.37840
Spleen (n = 5)	Mean Weight (g)	0.74700	0.84560	0.74560	0.91000
	Mean % Body	0.17333	0.18777	0.16988	0.19573
Testes (n = 10)	Mean Weight (g)	3.43	3.36	3.50	3.47
	Mean % Body	0.82614	0.78212	0.78701	0.78876
Thymus (n = 5)	Mean Weight (g)	0.26860	0.33920	0.33340	0.29420
	Mean % Body	0.06193	0.07543	0.07644	0.06293
Thyroid Gland (n = 5)	Mean Weight (g)	0.2380	0.02460	0.02840	0.02400
	Mean % Body	0.00550	0.00548	0.00648	0.00513

Dunnett's test: No statistically significant weight differences noted between treated groups and controls.

 

Table 9: Summary of Female Body/Organ Weights

Organ		Dose Group (mg/kg/day) n = 10
		0	100	300	1000
Final Body Weight (n = 10)	Mean Weight (g)	297.6	302.0	302.0	302.0
Adrenal Glands (n = 5)	Mean Weight (g)	0.08740	0.09440	0.09200	0.09280
	Mean % Body	0.03114	0.03150	0.03013	0.03166
Brain (n = 5)	Mean Weight (g)	1.93	2.02	1.98	1.95
	Mean % Body	0.68916	0.67280	0.66057	0.67211
Heart (n = 5)	Mean Weight (g)	0.96380	0.99780	0.96700	1.05
	Mean % Body	0.34367	0.33247	0.32203	0.35673
Kidneys (n = 5)	Mean Weight (g)	1.97	2.13	2.10	2.18
	Mean % Body	0.70297	0.71120	0.69474	0.74038
Liver (n = 5)	Mean Weight (g)	9.34	9.29	10.07	9.74
	Mean % Body	3.33	3.10	3.34	3.31
Ovaries (n = 5)	Mean Weight (g)	0.17900	0.22650#	0.21900	0.21320
	Mean % Body	0.06380	0.07521	0.07289	0.07279
Spleen (n = 5)	Mean Weight (g)	0.67280	0.65440	0.72060	0.65580
	Mean % Body	0.23993	0.21818	0.23827	0.22277
Thymus (n = 5)	Mean Weight (g)	0.22160	0.22260	0.31440	0.23500
	Mean % Body	0.07896	0.07465	0.10615	0.07945
Thyroid Gland (n = 5)	Mean Weight (g)	0.01820	0.01700	0.01860	0.01620
	Mean % Body	0.00649	0.00569	0.00613	0.00550
Uterus (n = 5)	Mean Weight (g)	0.79540	0.74700	0.70380	0.71460
	Mean % Body	0.28343	0.25007	0.23378	0.24407

# Significant in the Dunn’s test at 5%

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level was determined to be 1000 mg/kg bw/day since the test material did not induce any effects on systemic toxicity or reproductive toxicity at this dose.

Executive summary:

The reproductive toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female animals received daily doses of test material at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day for 2 weeks before mating, during mating and, for the females, throughout gestation until day 5 post partum. During the study animals were observed for mortality and clinical effects, and bodyweights and food consumption were recorded. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post partum. The total litter size and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 post partum. A functional observational battery was performed and blood biochemistry and haematology parameters were examined in parental animals. The males were sacrificed after completion of the mating period and seminology evaluations were performed. Dams were sacrificed on day 6 post partum. Body weights and selected organ weights were recorded and a complete macroscopic post mortem examination was performed with particular attention paid to the reproductive organs. Pups, including those found dead before study termination, were also submitted for macroscopic post mortem examination.

There were no unscheduled deaths in parental animals that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions which were considered to be due to a gavage trauma. There were no treatment-related effects on mating and fertility data or on body weight, food consumption, functional observation battery, blood biochemistry or haematology parameters and there were no treatment-related effects on the seminology parameters evaluated. Furthermore, there were no treatment-related findings noted at necropsy.

There were no treatment-related effects on the distribution of pups found dead and/or cannibalised and there were no treatment-related findings at pup examinations. Furthermore, there were no toxicologically significant effects on live birth, viability and lactation indexes. Although there was a tendency towards a decrease in mean body weight and mean body weight changes in pups at 1000 mg/kg/day, this finding was considered to be test material-related but of minor toxicological significance.

Therefore, under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day, the highest dose tested, both in terms of systemic toxicity of the parental animals and in terms of reproductive toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted under GLP and in accordance with a standardised guideline; the quality of the database is therefore high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female animals received daily doses of test material at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day for 2 weeks before mating, during mating and, for the females, throughout gestation until day 5 post partum. During the study animals were observed for mortality and clinical effects, and bodyweights and food consumption were recorded. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post partum. The total litter size and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 post partum. A functional observational battery was performed and blood biochemistry and haematology parameters were examined in parental animals. The males were sacrificed after completion of the mating period and seminology evaluations were performed. Dams were sacrificed on day 6 post partum. Body weights and selected organ weights were recorded and a complete macroscopic post mortem examination was performed with particular attention paid to the reproductive organs. Pups, including those found dead before study termination, were also submitted for macroscopic post mortem examination.

There were no unscheduled deaths in parental animals that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions which were considered to be due to a gavage trauma. There were no treatment-related effects on mating and fertility data or on body weight, food consumption, functional observation battery, blood biochemistry or haematology parameters and there were no treatment-related effects on the seminology parameters evaluated. Furthermore, there were no treatment-related findings noted at necropsy.

There were no treatment-related effects on the distribution of pups found dead and/or cannibalised and there were no treatment-related findings at pup examinations. Furthermore, there were no toxicologically significant effects on live birth, viability and lactation indexes. Although there was a tendency towards a decrease in mean body weight and mean body weight changes in pups at 1000 mg/kg/day, this finding was considered to be test material-related but of minor toxicological significance.

Therefore, under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day, the highest dose tested, both in terms of systemic toxicity of the parental animals and in terms of reproductive toxicity.

Short description of key information:

NOAEL (reproductive toxicity) = 1000 mg/kg bw/day (rat), OECD 422, Spézia (2013)

Justification for selection of Effect on fertility via oral route:

Only one study is available.

Effects on developmental toxicity

Description of key information

NOAEL (maternal toxicity) = 1000 mg/kg bw/day; NOAEL (developmental toxicity) = 1000 mg/kg bw/day (rat), OECD 422, Spézia (2013)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 February 2013 to 6 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl CD®(SD) IGS BR
- Age at study initiation: 10 weeks old (males); 9 weeks old (females)
- Weight at study initiation: 323 - 409 g (males); 204 - 277 g (females)
- Housing: Individually housed (except during mating) in polycarbonate cages with stainless steel lids and containing autoclaved sawdust.
- Diet: ad libitum
- Water: filtered tap water (0.22 µm filter), ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

drinking water treated by reverse osmosis

Details on exposure:

PREPARATION AND ADMINISTRATION OF DOSING SOLUTIONS
Test material was formulated in vehicle at the required concentrations. The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight.
A constant dose volume of 10 mL/kg/day was used.
Control animals (group 1) received vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test material in the dose formulations was analysed by GC-FID.

- Instrumentation: Varian Gas Chromatography system with FID detector
- Chromatographic conditions:
Column: HP-ULTRA (Agilent) 50 m x 0.32 mm (0.52 µm)
Mobile phase: hydrogen
Sensitivity: 12
Column temperature: from 70°C to 300°C at 10°C/min
Constant flow rate: 2 mL/min
Split rate: 10
Software: Empower 2 (Waters)
Detector temperature: 300°C
Injector temperature: 250°C
Injected volume: 2 µL
Detector gas flows: 30 mL/min for make up; 30 mL/min for hydrogen and 300 µL/min for air
Retention time: 8.4 min (test material); 8.8 min (internal standard)

Standard solutions of test material were prepared. An internal standard (1,4 diisopropylbenzene) was used to correct the injection variability.

- Preparation of Standard Solutions
Stock solutions of test material in diluent were prepared at 0.2, 0.02 and 0.0015 mg/mL.

- Preparation of Internal Standard
A solution of 1,4-diisopropylbenzene in methanol was prepared at 10 µg/mL.

ANALYTICAL METHOD
1 mL of dose formulation was diluted with diluent to reach the nominal concentration for injection. The diluted samples were analysed by GC-FID, bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions. The internal standard was used to correct the injection variability.

ANALYSIS OF DOSE FORMULATIONS
The test material concentrations in the administered dose formulations analysed in weeks 1, 4 and 6 remained within an acceptable range of -4.4 to +8.69 % when compared to the nominal values. Test material was not detected in the control samples.

Details on mating procedure:

Females were paired with males from the same dose level group. One female was placed with one male, in the latter's cage, during the night. Sibling pairings were avoided.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated day 0 post coitum.
Each female was placed with the same male until mating occurred or 14 days had elapsed.

Duration of treatment / exposure:

Males received dose formulations for 2 weeks before pairing, during the 2 week pairing period, until sacrifice (at least 5 weeks in total). Females received dose formulations for 2 weeks before mating, during the 2 week pairing period, during gestation, and during lactation until day 5 post partum inclusive (or until sacrifice).

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the selected dose levels were based on results of previous studies 2 acute oral studies where the LD50 values were > 5000 mg/kg and a 2-week preliminary toxicity study, by the oral route, in rats in which no animals died at dose levels up to 1000 mg/kg bw/day, however, some clinical signs were noted.
- Rationale for animal assignment: during the acclimation period, the required number of animals was selected according to body weight and/or clinical condition. The animals were allocated to groups (by sex) according to a computerised stratification procedure based on body weight, so that the average body weight of each group was similar.

Therefore, 1000 mg/kg/day was selected as the high dose-level for this study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and morbidity was checked once daily before the treatment period and at least twice a day during the treatment period. General clinical observations were performed once a day after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and weekly thereafter, observations were performed on all animals outside the home cage in a standard arena.
- Observations included (but were not limited to): changes in fur, skin, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the first day of treatment and weekly thereafter until sacrifice. Females were weighed on the first day of treatment then weekly until mated (or until sacrifice) and on days 0, 7, 14 and 20 post coital and days 1 and 5 post partum.
Bodyweight gain was calculated for selected intervals and for the overall study.

FOOD CONSUMPTION: Yes
- Food consumption by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period. The measures coincided with body weight measurements.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 post partum.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper at the next filling.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 6
> Macroscopic examination: A complete macroscopic examination was performed. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities/muscles with their associated organs and tissues and the neck with its associated organs and tissues. All gross lesions were recorded and preserved.
> Organ Weight: The body weight of each animal sacrificed as scheduled was recorded before sacrifice.The wet weights of the following were recorded as soon as possible after dissection: adrenals, brain (including medulla/pons cerebellar and cereebral cortex), epidiymides, heart, kidneys, liver, ovaries (with oviducts), prostate, seminal vesicles, spleen, testes, thymus, thyroids with parathyroids and uterus (horns and cervix).
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
> Histopathology: All tissues listed below were from the first five sacrificed as scheduled males and the first five females which delivered and were sacrificed on day 6 post partum of the control and high groups (groups 1 and 4) and for the female sacrificed prematurely:
adrenals, brain, cecum, colon, duodenum, epididymides, esophagus, gut-associated lymphoid tissue, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, ovaries, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord, spleen, sternum with marrow bone, stomach with forestomach, testes, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus and vagina.
Microscopic examination was also performed on all lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period.

OTHER: haematology parameters, blood biochemistry parameters and neurobehaviour were also tested.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. the number of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 post partum.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

The total litter size and numbers of each sex were recorded as soon as possible after birth. Any gross malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalised pups; clinical signs or abnormal behaviour were also recorded daily.
Furthermore, the body weight of each pup was recorded on days 1 and 5 post partum.

Pups found dead and pups sacrificed on day 5 post partum were carefully examined externally for gross external abnormalities. No macroscopic examination was performed and no tissues were preserved.

Statistics:

Data was compared by one-way analysis of variances and Dunnett test or by Fischer exact probability test. PathData software was used to perform the statistical analysis of organ weight data.

Indices:

The following indices were calculated: pre-implantation loss, post-implantation loss (manually calculated), mating index, fertility index, gestation index, live birth index, viability index and lactation index.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no unscheduled deaths that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions (piloerection, round back, swollen on left forelimb and chromorhinorrhea). At necropsy, a pouch was observed in the subcutaneous tissue of the left axillary region with a brown thick content which correlated histologically with acute inflammation. Inflammation with hemorrhage was seen in the larynx and particularly in surrounding tissues with edema and hemorrhage. There was no histological correlation with the perforation of the larynx noted at necropsy. Inflammation in tissues adjacent to the thymus was also observed. Additional changes consisted of a moderate hemorrhage in the left adrenal which correlated with a red discoloration at necropsy. Moderate increased apoptotic cell numbers was seen in the thymus along with decreased periarteriolar lymphoid sheaths (PALS) in the spleen. These observations in the lymphoid tissue were considered to be non specific, probably stress related. All these changes were suggestive of a gavage trauma due to the technical procedures (gavage error).
Ptyalism (hypersalivation) was recorded in males receiving 300 mg/kg/day (2/10 males) and in most males and females dosed at 1000 mg/kg/day during the premating period (9/10 males and 5/10 females) and in all females during the gestation and lactation period. This finding was considered to be related to the test material and a sign of discomfort but not an adverse effect.
A treatment-related effect was considered to be unlikely for the few other clinical signs as they are commonly observed in this species and strain or observed with no dose-level relationship.
There were no treatment-related effects in mean body weights and mean body weight changes. There were no biologically significant effects on mean food consumption associated with treatment with the test material.
There were no changes at necropsy which were indicative of a test material-related effect. Any changes observed were considered to be part of the normal background commonly seen in rats.
There were no changes in the mean organ weights which were suggestive of a test material-related effect. The small differences observed between groups were considered to reflect normal variations and were not considered to be due to treatment with the test material.
There were no changes at histopathological examination which were suggestive of a test material-related effect.
In the mandibular lymph nodes, presence of sinusal red blood cells was seen at a higher incidence in females given 1000 mg/kg/day than in controls. As this observation is commonly seen in rats, was observed unilaterally in 3/5 treated females and in the absence of a similar trend in males, any relationship with the test material was excluded.
Tubular basophilia was seen in the kidneys of 2/5 females given 1000 mg/kg/day but not in controls. As this observation was focal and unilateral, in the absence of a similar trend in males, any relationship with the test material was excluded.
Other changes observed were considered to be part of the normal background commonly seen in rats and without relationship with the test material.
Careful examination of testes, epididymides and ovaries did not reveal any treatment related changes.
There were no treatment related effects on functional observation battery observations or on the heamatology and blood chemistry parameters investigated.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 1000 mg/kg/day, there was a tendency towards an increased number of pups found dead (2.6% vs. 0.7% in controls) and cannibalised pups (3.2% vs 1.4% in controls). However, when compared with Historical Control Data (2009-2012), it is unclear whether this was a test material-related effect.
No treatment-related clinical signs were observed at pup examination.
There were no toxicologically significant effects on live birth, viability and lactation indexes associated with treatment with the test material.
At 1000 mg/kg/day, there was a tendency towards a decrease in mean pup body weight and mean body weight changes. This finding was considered to be test material-related but of minor toxicological significance as the difference was of low magnitude and remained within the range of 2009-2012 Historical Control Data.
There were no treatment-related effects on sex-ratio.
Furthermore, no macroscopically malformed pups were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

skeletal malformations

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Table 1: Delivery and Litter Data

Dose level (mg/kg bw/day)	0	100	300	1000
No. of pregnant females	10	9	10	10
No. of females which delivered	10	9	10	10
Mean duration of gestation (days)	21.3	21.3	21.6	21.5
Mean no. of corpora lutea	16.4	15.1	16.4	16.9
Mean no. of implantations	16.4	15.1	16.4	16.9
Mean pre-implantation loss (%)	0.0	0.0	0.0	0.0
Mean no. of pups delivered	14.5	14.2	15.0	15.4
Mean post-implantation loss (%)*	11.0	5.7	8.0	9.0
Viability index on day 4 post partum (%)	97.9	97.7	97.3	94.2

 * manually calculated, no statistics performed

Table 2: Mortality Data

Dose level (mg/kg bw/day)	0	100	300	1000
No. of pups found dead	1	3	3	4
No. of pups cannibalised	2	2	1	5
No. of pups delivered	145	128	150	154

 

Table 3: Clinical Signs and Gross External Examination

Dose level (mg/kg bw/day)	0	100	300	1000
No. of pups examined	145	128	150	154
Thin appearance	2	1		1
Cold to the touch	2	1		1
Necrosis (tail/hind limb)	1/1	4/4	3/3	2/2
Dehydration	1	1		4
Dyspnea		1
Heamatoma (head/back/abdomen)		4	4	8

Clinical signs/gross external observation

Table 4: Pup Viability

Dose level (mg/kg bw/day)	0	100	300	1000
Live birth index (%)	100	100	100	100
Pups dying, missing and/or cannibalised (days 1-4 p.p., %)	2.1	2.3	2.7	5.8
Viability index (day 4 p.p., %)	97.9	97.7	97.3	94.2
Lactation index (day 5 p.p., %)	100	98.4	100	100

p.p. = post partum

Table 5: Pup Body Weights (g)

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Day 1 p.p.	7.0	7.1	7.1	6.7	6.5	6.5	6.7	6.4
Day 5 p.p.	11.3	11.7	11.4	10.7	10.9	11.1	11.0	10.3
Body weight change (g)	+4.4	+4.6	+4.3	+4.0	+4.3	+4.6	+4.2	+4.0

p.p. = post partum

Table 6: Summary of Male Body/Organ Weights

Organ		Dose Group (mg/kg/day) n = 10
		0	100	300	1000
Final Body Weight (n = 10)	Mean Weight (g)	417.3	432.0	447.1	443.8
Adrenal Glands (n = 5)	Mean Weight (g)	0.07580	0.07700	0.07740	0.08400
	Mean % Body	0.01766	0.01708	0.01768	0.01799
Brain (n = 5)	Mean Weight (g)	2.09	20.3	2.08	2.09
	Mean % Body	0.48609	0.45047	0.47657	0.44759
Epididymides (n = 10)	Mean Weight (g)	1.44	1.40	1.41	1.46
	Mean % Body	0.34573	0.32473	0.31715	0.33084
Heart (n = 5)	Mean Weight (g)	134	1.39	1.36	1.45
	Mean % Body	0.31160	0.30831	0.31112	0.31067
Kidneys (n = 5)	Mean Weight (g)	3.03	3.13	3.21	3.34
	Mean % Body	0.70657	0.69605	0.73149	0.71510
Liver (n = 5)	Mean Weight (g)	10.94	11.48	10.82	12.04
	Mean % Body	2.53	2.55	2.46	2.58
Prostate (n = 5)	Mean Weight (g)	1.52	1.31	1.33	1.58
	Mean % Body	0.34868	0.28918	0.30124	0.33704
Seminal Vesicles (n = 5)	Mean Weight (g)	1.66	1.84	1.84	1.77
	Mean % Body	0.38802	0.40978	0.42236	0.37840
Spleen (n = 5)	Mean Weight (g)	0.74700	0.84560	0.74560	0.91000
	Mean % Body	0.17333	0.18777	0.16988	0.19573
Testes (n = 10)	Mean Weight (g)	3.43	3.36	3.50	3.47
	Mean % Body	0.82614	0.78212	0.78701	0.78876
Thymus (n = 5)	Mean Weight (g)	0.26860	0.33920	0.33340	0.29420
	Mean % Body	0.06193	0.07543	0.07644	0.06293
Thyroid Gland (n = 5)	Mean Weight (g)	0.2380	0.02460	0.02840	0.02400
	Mean % Body	0.00550	0.00548	0.00648	0.00513

Dunnett's Test: No statistically significant weight differences noted between treated groups and controls.

 

Table 7: Summary of Female Body/Organ Weights

Organ		Dose Group (mg/kg/day) n = 10
		0	100	300	1000
Final Body Weight (n = 10)	Mean Weight (g)	297.6	302.0	302.0	302.0
Adrenal Glands (n = 5)	Mean Weight (g)	0.08740	0.09440	0.09200	0.09280
	Mean % Body	0.03114	0.03150	0.03013	0.03166
Brain (n = 5)	Mean Weight (g)	1.93	2.02	1.98	1.95
	Mean % Body	0.68916	0.67280	0.66057	0.67211
Heart (n = 5)	Mean Weight (g)	0.96380	0.99780	0.96700	1.05
	Mean % Body	0.34367	0.33247	0.32203	0.35673
Kidneys (n = 5)	Mean Weight (g)	1.97	2.13	2.10	2.18
	Mean % Body	0.70297	0.71120	0.69474	0.74038
Liver (n = 5)	Mean Weight (g)	9.34	9.29	10.07	9.74
	Mean % Body	3.33	3.10	3.34	3.31
Ovaries (n = 5)	Mean Weight (g)	0.17900	0.22650#	0.21900	0.21320
	Mean % Body	0.06380	0.07521	0.07289	0.07279
Spleen (n = 5)	Mean Weight (g)	0.67280	0.65440	0.72060	0.65580
	Mean % Body	0.23993	0.21818	0.23827	0.22277
Thymus (n = 5)	Mean Weight (g)	0.22160	0.22260	0.31440	0.23500
	Mean % Body	0.07896	0.07465	0.10615	0.07945
Thyroid Gland (n = 5)	Mean Weight (g)	0.01820	0.01700	0.01860	0.01620
	Mean % Body	0.00649	0.00569	0.00613	0.00550
Uterus (n = 5)	Mean Weight (g)	0.79540	0.74700	0.70380	0.71460
	Mean % Body	0.28343	0.25007	0.23378	0.24407

# Significant in the Dunn’s test at 5%

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level was determined to be 1000 mg/kg bw/day for both maternal toxicity and developmental toxicity based on the absence of findings at this level.

Executive summary:

The developmental toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female animals received daily doses of test material at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day for 2 weeks before mating, during mating and, for the females, throughout gestation until day 5 post partum. During the study animals were observed for mortality and clinical effects, and bodyweights and food consumption were recorded. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post partum. The total litter size and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 post partum. A functional observational battery was performed and blood biochemistry and haematology parameters were examined in parental animals. Dams were sacrificed on day 6 post partum. Body weights and selected organ weights were recorded and a complete macroscopic post mortem examination was performed. Pups, including those found dead before study termination, were also submitted for macroscopic post mortem examination.

There were no unscheduled deaths in parental animals that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions which were considered to be due to a gavage trauma. There were no treatment-related effects on body weight, food consumption, functional observation battery, blood biochemistry or haematology parameters. Furthermore, there were no treatment-related findings noted at necropsy.

There were no treatment-related effects on the distribution of pups found dead and/or cannibalised and there were no treatment-related findings at pup examinations. Furthermore, there were no toxicologically significant effects on live birth, viability and lactation indexes. Although there was a tendency towards a decrease in mean body weight and mean body weight changes in pups at 1000 mg/kg/day, this finding was considered to be test material-related but of minor toxicological significance.

Therefore, under the conditions of the study the No Observed Adverse Effect Level was determined to be 1000 mg/kg bw/day for both maternal toxicity and developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted under GLP and in accordance with a standardised guideline; the quality of the database is considered to be fair since the study performed is a screening study for developmental toxicity.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The developmental toxicity of the test material was determined in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female animals received daily doses of test material at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/day for 2 weeks before mating, during mating and, for the females, throughout gestation until day 5 post partum. During the study animals were observed for mortality and clinical effects, and bodyweights and food consumption were recorded. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post partum. The total litter size and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 post partum. A functional observational battery was performed and blood biochemistry and haematology parameters were examined in parental animals. Dams were sacrificed on day 6 post partum. Body weights and selected organ weights were recorded and a complete macroscopic post mortem examination was performed. Pups, including those found dead before study termination, were also submitted for macroscopic post mortem examination.

There were no unscheduled deaths in parental animals that could be attributed to treatment during the study. In the 100 mg/kg/day group, one female was prematurely sacrificed on day 0 post coitum due to its poor clinical conditions which were considered to be due to a gavage trauma. There were no treatment-related effects on body weight, food consumption, functional observation battery, blood biochemistry or haematology parameters. Furthermore, there were no treatment-related findings noted at necropsy.

There were no treatment-related effects on the distribution of pups found dead and/or cannibalised and there were no treatment-related findings at pup examinations. Furthermore, there were no toxicologically significant effects on live birth, viability and lactation indexes. Although there was a tendency towards a decrease in mean body weight and mean body weight changes in pups at 1000 mg/kg/day, this finding was considered to be test material-related but of minor toxicological significance.

Therefore, under the conditions of the study the No Observed Adverse Effect Level was determined to be 1000 mg/kg bw/day for both maternal toxicity and developmental toxicity.

Justification for selection of Effect on developmental toxicity: via oral route:

Only one study is available.

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for reproductive or developmental toxicity.