Octanoic acid, 2-phenoxyethyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL systemic P1 (oral, rat, OECD 421) = 1000 mg/kg bw/day

NOAEL fertility P1 (oral, rat, OECD 421) = 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Mar - 06 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted in Jul 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

2000

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

Version / remarks:

2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 14 - 15 weeks
- Weight at study initiation: Males: 350 - 473 g (range); females: 223 - 281 g (range)
- Housing: animals were housed in groups of 5 animals/sex/cage in IVC cages (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Mar 2017 To: 21 May 2017

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of PEG 400 and further vortexing it for 2-3 minutes. The test item and control formulations were prepared once in 10 days. The formulations were stored at ambient temperature, protected from light. Homogeneity of the test item in the vehicle was maintained thoroughly (using magnetic stirrer) before every dose administration.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on the test item’s characteristics and previous 28-day toxicity study (NOTOX Project 497712)
- Concentration in vehicle: low-dose: 20 mg/mL; mid-dose: 60 mg/mL; high-dose: 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: S 71206885543, S 7263585626

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the low-dose dose group was between 100.0% and 99.1% of the nominal value, between 98.7% and 99.2% for the mid-dose dose group and between 98.4% and 99.1% of the nominal value for high-dose dose group.
The mean recoveries observed in the low-dose, mid-dose and high-dose groups were 99.6%, 99.3%, and 99.0% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within the acceptance criterion of ± 10%.

Duration of treatment / exposure:

Males: 28 days (during 14 days of pre-mating and maximum 14 days of mating until 28 days were completed)
Females: 63 days (during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 12)

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the previous 28-day toxicity study (NOTOX Project 497712, please refer to 7.5.1) performed with same test item, and also in consultation with the sponsor the doses 100, 300 and 1000 mg/kg bw/day were selected.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations of all animals were made at least once a day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on prenatal day (PND) 4 and 13 along with pups. All animals were weighed at termination.

FOOD CONSUMPTION:
- Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and during the post-mating period in males.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select females with regular estrous cyclicity for the study. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births and runts, and presence of gross abnormalities as soon as possible after delivery; anogenital distance (AGD) was measurend on PND 0 and numbers of nipples/areolae in male pups was counted on PND 12. Litter weight was determined on day 0, 4 and day 13 post-partum. Blood samples from the day-13 pups were assessed for serum levels for thyroid hormones (T4).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 30 using an anaesthesia (ketamine/xylazin).
- Maternal animals: Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. Dams were sacrificed on post-natal day 13 using an anaesthesia (ketamine/xylazin).

GROSS NECROPSY
- Gross necropsy consisted of a careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
- A full histopathology was carried out on the preserved organs and tissues of all male and female animals of the control and high dose groups which were sacrificed at the end of the treatment period and of all animals which died during the study. The following tissues were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol: all gross lesions, epididymides, ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix and vagina, and thyroid gland.
- The wet weight of the reproductive organs of all sacrificed adult males and all lactating females from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded.The following organs were weighed at necropsy: testes, epididymides, prostate, seminal vesicles and coagulating glands, Cowper’s gland, levator ani + bulbocavernosus muscle complex, glans penis, uterus with cervix, ovaries and thyroid gland (after fixation).

Postmortem examinations (offspring):

SACRIFICE
- All surviving pups were killed by cervical dislocation on PND 13.

GROSS NECROPSY
- Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Thyroid gland from 1 pup/sex/litter/ group sacrificed on PND 13 was preserved for the potential histopathological examination.
- Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) were weighed.

Statistics:

A statistical assessment of the results of the body weight, food consumption and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100

Offspring viability indices:

Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Control: salivation was noted in 1/10 males on mating day (MD) 10, in 1/10 males on MD 9 and 11, in 1/10 females on gestation day (GD) 7; 3/10 males (1/10 on MD 1-10, 1/10 on MD 1-5 and 1/10 on MD 11-12) and 2/10 females (1/10 on GD 0-8, GD 11 and GD 13-15, and 1/10 on PND 0) had diarrhoea; moving the bedding was noted for 3/10 females (1/10 on GD 5-9 and GD 13-15, 1/10 on GD 7-8 and 1/10 on PND 1) and abnormal breathing was noted in 1/10 females on PND 1
100 mg/kg bw/day: moving the bedding was observed for 1/10 males on premating day (PMD) 10 and for 6/10 females (1/10 on PMD 9-10, 1/10 on PMD 9, 1/10 on GD 19-20, 1/10 on GD 18, 1/10 on GD10 and 1/10 on GD 5, 18 and 20); salivation on GD 6, nasal discharge on PND 9 and 10 and regurgitation of test item on PMD 10 was noted for 1/10 females
300 mg/kg bw/day: 2/10 males (1/10 on MD 9-10, 1/10 on MD 3, MD 5-10 and 14) and 3/10 females (1/10 on PMD 10, 1/10 on GD 20-21 and 1/10 on GD 5) showed salivation; diarrhoea was observed for 3/10 males (1/10 on MD 4-12 and 14 and in 2/10 on MD 12 and 14-15); moving the bedding was noted in all males and in 9/10 females during the premating period, respectively; regurgitation of test item was observed for 1/10 males on PMD 10; 1/10 females showed alopecia on PND 13 and abnormal breathing on PMD 8, respectively
1000 mg/kg bw/day: Salivation was noted in 2/10 males and in 3/10 females (1/10 on GD 0-4, 1/10 on PMD 10 and 1/10 on GD 10 and 12); diarrhoea occurred in 1/10 males on MD 12 and 14-15; all males and all females showed moving the bedding during premating, mating and gestation period, respectively; nasal discharge on PND 4 and scratch on left hindlimb on PND 7-13 were observed for 1/10 females, respectively.

Isolated incidences of alopecia on various body parts, slight to moderate red nasal discharge, abnormal breathing, scratch and regurgitation of test item were observed on few occasions in very few female animals in all groups including control group and considered to be incidental in nature.
Moving the bedding was observed transiently in few males and females of the control (only female), 100 mg/kg bw/day group and almost all animals of the 300 and 1000 mg/kg bw/day group. The clinical signs salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

Please refer to Table 1 under "Any other information on results incl. tables".

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Control: 1/10 females was found dead on premating day 9 and 1/10 females was found dead on gestation day (GD4), respectively. No specific clinical signs were observed before death in both females.
300 mg/kg bw/day: 1/10 females was found dead on premating day 8. Clinical signs observed before death was abnormal breathing on premating day 8.

Histopathologically, the cause of death of the control females was not evident. The cause of death of the female in the 300 mg/kg bw/day group was considered to be due to accidental aspiration of the vehicle and/or the test material, i.e. amorphous material was noted in the lungs. Although no histological correlate was found in the lung section, the gross lesion of fluid filled lungs indicate death due to accidental aspiration.

Please refer to Table 2 under "Any other information on results incl. tables".

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

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:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Gross lesions and histological alterations were unremarkable and not related to treatment with the test item. In non-mating animals, there were no histological lesions.
No histopathological findings were observed in the animals that died during the study period.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The test substance had no biologically significant effect on the estrous cycle analyzed during 2 weeks premating period after the first test substance administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed in one animal in the 300 mg/kg bw/day group. As this effect was observed in just one animal and due to lack of dose dependency, it was considered as biological variation and not related to the treatment with the test substance.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test substance related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared with the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related and adverse effects observed up to and including the highest tested dose level.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related and adverse effects observed up to and including the highest tested dose level.

Key result

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects on the survival of the pups from PND 1 through PND 13 in the dose groups when compared to the control group. However, mean mortality of pups between PND 0 and PND 4 in the 100 mg/kg bw/day group was (0.89%) compared to the control group (1.39%). This outcome did not achieve statistical significance and was attributed to missing one single pup of one single dam in the 100 mg/kg bw/day group on PND 1. In control group also, one pup observed missing on PND 1. Thus, due to lack of dose dependency in absence of effect in the 300 and 1000 mg/kg bw/day group, this effect on pup survival in the 100 mg/kg bw/day group was considered as incidental and not related to the treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: Significantly lower mean pup body weights on PND O were measured in males (10% decrease) and females (7% decrease) when compared with the control. There was no significant difference in mean pup weight of females and males combined between the control and treatment groups. Furthermore, a higher mean number of pups per litter was noted for this dose group, which should be taken into account since these result in lower individual weights of each pup at birth. The mean body weights on PND 13 formales and females, calculated separately and combined, were in the same range when compared with the control animals and were thereby shown to be transient. In addition, the mean litter weight for the 300 mg/kg bw/day group was comparable to the control group value. The effect on mean pup body weight in females and males separately noted on PND 0 were not considered to be toxicologically relevant.

1000 mg/kg bw/day: A significant reduction of the mean pup body weight was noted in males (7% decrease) and females (9% decrease) on PND O when compared with the control, respectively. There was no significant difference in mean pup weight of females and males combined between the control and treatment groups. The mean body weights on PND 13 formales and females, calculated separately and combined, were in the same range when compared with the control animals and were thereby shown to be transient. In addition, the mean litter weight for the 1000 mg/kg bw/day group was comparable to the control group value. The effect on mean pup body weight in females and males separately noted on PND 0 were not considered to be toxicologically relevant.

Please refer to Table 3 under "Any other information on results incl. tables".

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:

no effects observed

Description (incidence and severity):

No test substance-related effect of toxicological relevance was observed in male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

In males, no test substance related effect of toxicological relevance was observed on nipple retention in the pups of any of the groups. However, statistically significant lower absolute and relative anogenital distance (AGD), pup weight and cube root of pup weight of male pups was observed in the 300 and 1000 mg/kg bw/day group when compared with the controls.
This effect on anogenital distance in the 300 and 1000 mg/kg bw/day group was statistically significant but not considered to be biologically or toxicologically relevant due to the high variation in inherent the methodology of measuring the AGD.

In females, no statistically significant effect on anogenital distance and relative anogenital distance was observed in any treatment groups when compared with the controls.

Please refer to Table 4 under "Any other information on results incl. tables".

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significant higher (increase of 40%) group mean thyroid weights were observed in pups in the 1000 mg/kg bw/day group when compared with the controls.

The analysis of the mean thyroxine (T4) on PND 13 revealed no treatment-related effect in all dose groups in males and females, respectively, when compared with the control group. Therefore and since the increase of the thyroid weight was minimal (%), this effect in male thyroid weight was not considered to be adverse.

Please refer to Table 5 under "Any other information on results incl. tables".

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pup External Findings
No test substance related gross external abnormalities were observed on PND 0 in the pups of any of the groups.

Litter data
There were no test substance treatment-related effects on litter data including total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13.

Pre- and Post-natal data
There were no test substance treatment-related effects on the number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related, adverse effects observed at and including the highest tested dose level of 1000 mg/kg bw/day.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Clinical Observations in males and females

Clinical Finding	Control	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Males
Total number of animals examined	10	10	10	10
salivation	2	-	2	2
diarrhoea	3	-	3	1
moving the bedding	-	1	10	10
regurgitation of test item	-	-	1	-
Females
Total number of animals examined	10	10	10	10
salivation	1	1	3	3
diarrhoea	2	-	-	-
moving the bedding	3	4	9	10
alopecia	-	-	1
nasal discharge	-	1	-	1
abnormal breathing	1	-	1	-
scratch	-	-	-	1
regurgitation of test item	-	1	-	-

Table 2: Mortality: Summary

Group	Gender		Day and cause of death
	Male	Female
Control	0/10	2/10	found dead (GD 4 and PMD 9)
100 mg/kg bw/day	0/10	0/10	-
300 mg/kg bw/day	0/10	1/10	found dead (PMD 8)
1000 mg/kg bw/day	0/10	0/10	-

GD: Gestation day

PMD: Premating day

Table 3: Pup mean weight (g) on PND 0 and PND 13

Group		Male Mean Pup Weight (g) PND 0	Female Mean Pup Weight (g) PND 0	Mean Pup Weight (g) PND 0	Total litter weight (g) PND 0	Male Mean Pup Weight (g) PND 13	Female Mean Pup Weight (g) PND 13	Mean Pup Weight (g) PND 13	Total litter weight (g) PND 13
Control	Mean	6.77	6.43	6.65	79.80	28,48	27,95	28.88	289.49
	SD	0.72	0.77	0.66	11.41	4,54	4,41	4.55	23.17
	N	43	54	8	8	42	40	8	8
100 mg/kg bw/day	Mean	6.82	6.41	6.63	78.40	30,47	29,48	30.06	300.53
	SD	0.61	0.61	0.58	12.95	2,10	1,83	1.96	36.68
	N	47	48	8	8	46	33	8	8
300 mg/kg bw/day	Mean	6.05***	5.96**	6.09	83.34	25,27	25,43	25.92	307.13
	SD	0.76	0.89	0.80	14.00	3,22	4,22	4.00	28.00
	N	66	59	9	9	66	44	9	9
1000 mg/kg bw/day	Mean	6.34**	5.87***	6.17	73.29	29,54	26,64	28.98	290.13
	SD	0.59	0.59	0.52	12.36	3,47	3,89	3.81	34.78
	N	64	56	10	10	64	38	20	10

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 4: Results of F1-offspring parameters

		Male Pups					Female Pups
Group		Pup Weight (g)	Cube Root of Pup Weight	Anogenital Distance (mm) of Pups	Relative Anogenital Distance Pups	Pup nipple retention (N) on PND 12	Pup Weight (g)	Cube Root of Pup Weight	Anogenital Distance (mm) of Pups	Relative Anogenital Distance Pups
Control	Mean	6.77	1.89	2.62	1.38	0.12	6.43	1.86	0.75	0.40
	SD	0.72	0.07	0.36	0.18	0.40	0.77	0.08	0.30	0.15
	N	43	43	43	43	42	54	54	54	54
100 mg/kg bw/day	Mean	6.82	1.90	2.48	1.31	0.02	6.41	1.86	0.81	0.44
	SD	0.61	0.06	0.33	0.16	0.15	0.61	0.06	0.34	0.19
	N	47	47	47	47	46	48	48	47	47
300 mg/kg bw/day	Mean	6.05***	1.82***	2.35**	1.29*	0.12	5.96**	1.81**	0.77	0.42
	SD	0.76	0.08	0.43	0.20	0.37	0.89	0.09	0.24	0.13
	N	66	66	66	66	66	59	59	59	59
1000 mg/kg bw/day	Mean	6.34**	1.85*	2.38**	1.28*	0.09	5.87***	1.80***	0.84	0.47
	SD	0.59	0.06	0.43	0.20	0.34	0.59	0.06	0.34	0.19
	N	64	64	64	64	64	56	56	56	56

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 5: Mean Organ Weight Thyroid Gland (Post Fixation) Pups - Day 13

Group		Male Pups - Thyroid/Parathyroid glands (g)	Female Pups - Thyroid/Parathyroid glands (g)
Control	Mean	0.0043	0.0049
	SD	0.0008	0.0016
	N	8	8
100 mg/kg bw/day	Mean	0.0057	0.0061
	SD	0.0015	0.0012
	N	8	8
300 mg/kg bw/day	Mean	0.0056	0.0051
	SD	0.0013	0.0005
	N	9	9
1000 mg/kg bw/day	Mean	0.0061*	0.0060
	SD	0.0014	0.0014
	N	10	10

Asterisks indicate statistically significant differences to reference item group, with* p<0.05, ** p<0.01 and *** p<0.001.

Conclusions:

The test substance had no effect on reproductive performance and no effect on development in the offspring. Therefore the NOAEL for systemic and for reproduction and development was considered to be 1000 mg/kg bw/day.

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 available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reliable data is available for the test substance which was tested in a reproduction/developmental toxicity screening study according to OECD guideline 421 and under GLP (BVL, 2017). 10 Wistar rats per sex and dose were treated via gavage with the test substance at doses of 100, 300 and 1000 mg/kg bw/day, respectively. The control group received the vehicle polyethylene glycol. Males were treated for 28 days (during 14 days of pre-mating and maximum 14 days of mating). Females were treated for 63 days (during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 12).

In parental animals no toxicologically relevant clinical signs were observed. Salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to the test item administration rather than a systemic adverse effect and has no toxicological relevance. In both males and females, there was no treatment-related effect on body weight and body weight gain during the study period. The food consumption was correlated to the body weight gain. During the treatment period of this study, 1/10 control females was found dead on gestation day (GD) 4, and 1/10 control females was found dead on premating day 9. One (1/10) mid-dose female was found dead on premating day 8. The cause of death of one of the control and the mid-dose female was not evident. The cause of death in the 2nd control female was considered to be due to accidental aspiration of the vehicle, as amorphous material was noted in the lungs. Although no histological correlate was found in the lung section, the gross lesion of fluid filled lungs indicates death due to accidental aspiration. Statistically significantly higher absolute and relative (to body weight) glans penis weight was observed in high-dose group males when compared with the controls. This increase was not considered to be adverse since 2/10 animals had a distinct higher organ weight than the other animals of the high-dose group leading to a higher mean organ weight. There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared with the control group. However, a slightly reduced fertility index of 80% was observed in the low-dose group compared with 88.9% in the control group. This effect on fertility index was considered to be a biological variation and not related to the treatment.

Based on the results of this study, the NOAEL systemic was derived to be 1000 mg/kg bw/day and the NOAEL for reproductive performance was also derived to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

NOAEL systemic P1 (oral, rat, OECD 421) = 1000 mg/kg bw/day

NOAEL developmental toxicity F1 (oral, rat, OECD 421) = 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

Reliable data is available for the test substance which was tested in a reproduction/developmental toxicity screening study according to OECD guideline 421 and under GLP (BVL, 2017). 10 Wistar rats per sex and dose were treated via gavage with the test substance at doses of 100, 300 and 1000 mg/kg bw/day, respectively. The control group received the vehicle polyethylene glycol. Males were treated for 28 days (during 14 days of pre-mating and maximum 14 days of mating). Females were treated for 63 days (during 14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 12).

In parental animals no toxicologically relevant clinical signs were observed. Salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to the test item administration rather than a systemic adverse effect and has no toxicological relevance. In both males and females, there was no treatment-related effect on body weight and body weight gain during the study period. The food consumption was correlated to the body weight gain. During the treatment period of this study, 1/10 control females was found dead on gestation day (GD) 4, and 1/10 control females was found dead on premating day 9. One (1/10) mid-dose female was found dead on premating day 8. The cause of death of one of the control and the mid-dose female was not evident. The cause of death in the 2nd control female was considered to be due to accidental aspiration of the vehicle, as amorphous material was noted in the lungs. Although no histological correlate was found in the lung section, the gross lesion of fluid filled lungs indicates death due to accidental aspiration. Statistically significantly higher absolute and relative (to body weight) glans penis weight was observed in high-dose group males when compared with the controls. This increase was not considered to be adverse since 2/10 animals had a distinct higher organ weight than the other animals of the high-dose group leading to a higher mean organ weight.

Significantly lower mean pup body weights on PND 0 were measured in males (10% decrease) and females (7% decrease) at 300 mg/kg bw/day, when compared with the control. There was no significant difference in mean pup weight of females and males combined between the control and treatment groups. Furthermore, a higher mean number of pups per litter was noted for this dose group, which should be taken into account since these result in lower individual weights of each pup at birth. The mean body weights on PND 13 for males and females, calculated separately and combined, were in the same range when compared with the control animals and were thereby shown to be transient. In addition, the mean litter weight for the 300 mg/kg bw/day group was comparable to the control group value. The effect on mean pup body weight in females and males separately noted on PND 0 were not considered to be toxicologically relevant. A significant reduction of the mean pup body weight was noted in males (7% decrease) and females (9% decrease) at 1000 mg/kg bw/day on PND 0 when compared with the control, respectively. There was no significant difference in mean pup weight of females and males combined between the control and treatment groups. The mean body weights on PND 13 for males and females, calculated separately and combined, were in the same range when compared with the control animals and were thereby shown to be transient. In addition, the mean litter weight for the 1000 mg/kg bw/day group was comparable to the control group value. The effect on mean pup body weight in females and males separately noted on PND 0 were not considered to be toxicologically relevant.

In males, no treatment-related effect was observed on nipple retention in the pups of any of the groups. A significantly lower absolute and relative anogenital distance (AGD), pup weight and cube root of pup weight of male pups was observed in the 300 and 1000 mg/kg bw/day group when compared with the controls. This effect on AGD in the 300 and 1000 mg/kg bw/day group was statistically significant but not considered to be toxicologically relevant due to the high variation inherent in the methodology of measuring the AGD. Significantly higher group mean thyroid weights (approximately 30%) were observed in pups in the 1000 mg/kg bw/day group when compared with the controls. However, the increase in the thyroid weight was not dose-related and the standard deviation was relatively large in all the groups, indicating relatively high inter-individual differences. The effect in male thyroid weight was therefore not considered to be toxicologically relevant. The mean thyroxine (T4) value on PND 13 was comparable between the treatment groups and the control group. Other developmental parameters such as total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13, were not affected by the treatment with the test substance.

Based on the results of this study, the NOAEL systemic was derived to be 1000 mg/kg bw/day for parental animals and a NOAEL of 1000 mg/kg bw/day was derived for the offspring.

Justification for classification or non-classification

The available data on toxicity to reproduction does not meet the classification criteria according to Regulation (EC) 1272/2008. However, as no prenatal developmental toxicity study is available, the conclusion for classification is ´data lacking`.