2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, reaction products with phosphorus oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effect on reproductive performance as well as offspring was observed in the combined repeated dose and reproduction / developmental screening test.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 26, 2016 to March 24, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other assay used for intermediate effect derivation

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but considered not to adversely affect the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

other: OECD guidance document 43

Version / remarks:

The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

GLP compliance:

yes

Limit test:

no

Justification for study design:

- To obtain information on the possible toxic effects of the test substance following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (propylene glycol).
- The study included a reproductive/ developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance including gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 4 post-partum.

Specific details on test material used for the study:

Name: PARAD Substance 139
Batch number: JBLB0008S
Purity: 100%
Appearance: Clear colourless liquid
The test substance was formulated in the vehicle, as a clear solution at the appropriate concentrations according to the selected dose level and volume, in the Pharmacy of CiToxLAB Hungary Ltd.
Stability of the test substance in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to CiToxLAB study code 15/340-316AN).

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany)
Housing conditions: SPF at the supplier, standard laboratory conditions during the study
Number of animals: 51 male, 51 female rats, 4 groups. 12 animals/sex/group, with the exception of the High dose group, where 15 animals/sex/group was used. Animals originated from different units, to avoid brother/sister matings
Age of animals: young adult rats, at least 10 weeks old at starting and 12 weeks at mating
Body weight at the start: males: 362-422 g, females: 218-261 g
Acclimation period: at least 5 days
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-24.3 °C (target: 22 ± 3°C)
Relative humidity: 40 - 62% (target: 30 - 70%)
Ventilation: 15-20 air exchanges/hour
Food and water supply: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" and tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

The oral route was selected, as it is a possible route of exposure to the test substance in humans.

Details on mating procedure:

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 14 days. If the first 14 days of the mating was unsuccessful, then the female was paired for a second period with a proven male from the same dose group (1:1 mating).
A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

Analysis of test substance formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations and all concentrations. Sample analysis was performed on 3 occasions (with an additional occasion for the High dose (250 mg/ml) formulation) for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test substance. Description of the analytical method and the results of the formulation analysis are included in the Analytical report provided by the Analytical Laboratory of CiToxLAB Hungary Ltd.
Analysis of the test substance formulation samples of 1-250 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 days at room temperature.
The measured concentrations of the test substance evaluated for each test substance-dose group varied between 94 % and 107 % of the nominal contents. No test substance was detected in the control samples. These results were within the acceptable range (85% - 115%) and are acceptable for the study purposes. All samples were found to be homogeneous.

Duration of treatment / exposure:

- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and then euthanized and subjected to necropsy examination.
- Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. (Females showing no-evidence of copulation were sacrificed 25 days after the end of the mating period (two animals in the Control and two animals in the Mid dose, with no delivery).
- All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

Frequency of treatment:

daily on a 7 days/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 4 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights. (Dosing of both sexes began after at least 5 days of acclimation and 2 weeks before mating and continued up to the day before necropsy.)

Dose / conc.:

0 mg/kg bw/day

Remarks:

Concentration: 0 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

100 mg/kg bw/day

Remarks:

Concentration: 25 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

300 mg/kg bw/day

Remarks:

Concentration: 75 mg/mL
Dose volume: 4 mL/kg bw

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Concentration: 250 mg/mL
Dose volume: 4 mL/kg bw

No. of animals per sex per dose:

at least 12

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work including the results of a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 15/340-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
Based on the results from these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

Positive control:

-

Parental animals: Observations and examinations:

Refer to the study under repeated dose endpoint (section 7.5.1) for details on systemic parameter examinations.

Litter observations:

Observation of the delivery process, offspring and nursing instinct:
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition. Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0 or PND1) and on PND4, with accuracy of 0.01g. All litters were checked daily for the number of viable and dead pups. All pups were sacrified on PND4.

Postmortem examinations (parental animals):

- Macroscopic evaluation
Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the females as applicable.

- Organ weight measurements

At the time of termination, body weight and the weight of the following organs from all euthanized adult animals were determined:
- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Paired organs were weighed together except testes and epididymides, which were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

- Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution; all other organs in 10% buffered formalin solution. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Detailed histological examination was performed on all retained organs in the control and Hhigh dose groups and any macroscopic findings (abnormalities) observed in all animals. Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2 test). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA/ANCOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test or Kruskal-Wallis test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. For SMART evaluation, T-test was used.

Reproductive indices:

Male Mating Index % (Measure of male’s ability to mate): "Number of males with confirmed mating" /"Total number of males cohabited" "x100"

Female Mating Index % (Measure of female’s ability to mate): "Number of sperm-positive females" /"Total number of females cohabited" "x100"

Male Fertility Index % (Measure of male’s ability to produce sperm that can fertilise eggs): "Number of males impregnating a female" /"Total number of males cohabited" "x100"

Female Fertility Index % (Measure of female’s ability to become pregnant): "Number of pregnant females" /"Number of sperm-positive females" "x100"

Gestation Index % (Measure of pregnancy that provides at least one live pup): "Number of females with live born pups" /"Number of pregnant females" "x100"

Offspring viability indices:

Survival Index %: "Number of live pups (at designated time)" /"Number of pups born" "x100"

Pre-implantation mortality %: "Number of corpora lutea-Number of implantations" /"Number of corpora lutea" "x100"

Intrauterine mortality %: "Number of implantations-Number of liveborns" /"Number of implantations" "x100"

Total mortality %: "Number of implantations-Number of viable pups (PND4)" /"Number of implantations" "x100"

Sex ratio %: "Number of pups examined-Number of males" /"Number of pups examined" "x100"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance related effects were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels up to and including 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Refer to the RSS under repeated dose (section 7.5.1) for details on results

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive ability assessment and indices:
There were no differences between the control and test substance treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the test substance administration. The mating indices were normal in all groups (93-100%). The fertility index was 83, 100, 83 and 92% for males, and 83, 100, 83, 100% in females in the Control, Low, Mid and High dose groups, respectively. The gestation index was 100% in all groups.

Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 4 days of pairing (cohabitation). In one High dose female (ID 4507), the duration of the mating period was 14+4 days, with continuous dioestrus observed during the oestrus cycle, however after the new male was introduced after Day 14, this female successfully paired on Day 18 of the mating period (after 4 days). Therefore, the elongated mating period may have been partly due to the male animal (ID 4007), where this variation was regarded as individual, normal biological variability and without toxicological significance.

Mean pre-coital interval was 2.2, 2.5, 2.8 and 2.4 days in Control, Low, Mid and High dose groups, respectively.

Evaluation of the gestation, parturition and post-partal period:
There was no effect of treatment noted during gestation, parturition or the post-partal period. Delivery lasted more than three hours in one Low (100 mg/kg bw/day) and one High (1000 mg/kg bw/day) dose animal. These relatively long parturition durations were considered to be incidental, and not related to the treatment. Statistically shorter duration of pregnancy occured in the Low (100 mg/kg bw/day, p<0.01) and High (1000 mg/kg bw/day, p<0.05) groups, but all animals in the study had a pregnancy duration of 22 or 23 days, so all were considered normal. The statistical difference was considered incidental and unrelated to the treatment. The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups. There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, intrauterine, post-natal or total mortality values (litter mean and %) at up to and including 1000 mg/kg bw/day.

For details refer to 'any other information on results incl. tables'

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: no adverse effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day. Overall, there were no treatment-related effects on pup mortality. There were no treatment related effect on the viability of pups on PND0 and PND 4.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects considered adverse on the offspring weight or weight gain following administration of the test substance at 100, 300 or 1000 mg/kg bw/day to parental generation under the conditions of this study. When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were recorded.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Reproductive effects observed:

no

Summary of reproductive parameters, females

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead and preterminal euthanasia)	0	0	0	5
Number of mated animals	12	12	12	12*
Number of sperm positive females	12	12	12	12**
Number of females with corpora lutea,  but no implantation sites	2	0	2	0
Number of pregnant females	10	12	10	10
Number of pregnant females with live born(s)	10	12	10	10
Female mating index (%)	100	100	100	100
Female fertility index (%)	83	100	83	100
Female gestation index (%)	100	100	100	100

*One additional paired female animal (No. 4503) was found dead during the mating period, and therefore was excluded from further analysis.

** For two sperm positive females, mortality occurred during the early stages of pregnancy, therefore fertility index was calculated after these animals were excluded from sperm positive females (number of animals included for further calculations: 10.)

 

Summary of the intrauterine evaluation

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of evaluated females	12	12	12	10
Mean number of corpora lutea	13.00	15.83	14.25	14.60	NS
Pre-implantation mortality, mean	1.67	0.00	1.50	0.20	NS
Pre-implantation mortality (%), mean	18.33	0.00	16.67	1.05	NS
Mean number of implantations	11.33	15.83	12.75	14.40	NS
Number of evaluated litters	10	12	10	10
Intrauterine mortality, mean	0.40	0.67	0.50	0.50	NS
Intrauterine mortality (%), mean	4.81	4.05	3.20	5.64	NS
Post-natal mortality, mean	0.10	0.25	0.20	0.20	NS
Post-natal mortality (%), mean	0.71	1.47	1.21	3.33	NS
Total mortality, mean	0.50	0.92	0.70	0.70	NS
Total mortality (%), mean	5.52	5.44	4.35	7.84	NS
Duration of pregnancy (days)	22.50	22.00**	22.30	22.10*	DN
Number of pups born, mean	13.20	15.42	14.90	14.00	NS
Number of live born, mean	13.20	15.17	14.80	13.90	NS

 Notes: Mean values were rounded to two decimal places.

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

DN = Duncan's Multiple Range Test, NS = Non Significant

Summary of reproductive parameters, males

Parameters	Groups/Concentration (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000)
Number of treated animals	12	12	12	15
Number of preterminal death (found dead)	0	0	0	1
Number of mated animals	12	12	12	13
Number of infertile animals	0	0	0	1
Male mating index (%)	100	100	100	93
Male fertility index (%)	83	100	83	100

Note: Male fertility figures takes account only of males where the mated female survived to the end of gestation.

Conclusions:

Under the study conditions and in absence of adverse effects, the rat NOAEL for reproductive and development effects can be considered to be at 1000 mg/kg bw/day.

Executive summary:

A screening study was conducted to determine the reproductive and development toxicity of the test substance according to OECD guideline 422. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Three groups of 12 male and 12 female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. The males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and females for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, apart from systemic parameter examinations data was recorded reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals. Organ weight (including gonads), macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.Daily administration of the test substance at dose levels of 100 or 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters. Test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg/d) and clinical adverse effects were present in High dose females. There were no effects on surviving animals on body weight or clinical pathology. Test substance related effects in liver and kidney in the High dose group were considered to be an adaptive change and not an adverse effect of treatment. Further, no test substance-related microscopic changes were noted in the reproductive organs in any groups.

Under the study conditions, the rat NOAEL for reproductive and development effects was established at 1000 mg/kg bw/day and at 300 mg/kg bw/day for systemic effects (Hargitai, 2016).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening study was conducted to determine the reproductive and development toxicity of the test substance according to OECD guideline 422. The general systemic toxic potential of the substance to Wistar rats was assessed by daily oral administration (gavage). Three groups of 12 male and 12 female rats received the test substance at doses of 0, 100, 300 or 1000 mg/kg bw/day. The males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating), and females for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 days post-partum dosing). A similarly constituted control group received the vehicle, propylene glycol, at the same volume-dose. During the study, apart from systemic parameter examinations data was recorded reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals. Organ weight (including gonads), macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. Daily administration of the test substance at dose levels of 100 or 300 mg/kg bw/day did not result in adverse changes in clinical signs, neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry, or urinalysis parameters. Test substance related mortality (approximately 15% mortality) was observed in the High dose group (1000 mg/kg/d) and clinical adverse effects were present in High dose females. There were no effects on surviving animals on body weight or clinical pathology. Test substance related effects in liver and kidney in the High dose group were considered to be an adaptive change and not an adverse effect of treatment. Further, no test substance-related microscopic changes were noted in the reproductive organs in any groups.

Under the study conditions, the rat NOAEL for reproductive and development effects was established at 1000 mg/kg bw/day and at 300 mg/kg bw/day for systemic effects (Hargitai, 2016).

Mode of Action Analysis / Human Relevance Framework

None

Justification for classification or non-classification

Based on the results of a combined repeated dose toxicity and reproductive/developmental screening study (OECD 422) with the test substance, no classification is required for this endpoint according to CLP (1272/2008) criteria.​