Bis(2-(2-butoxyethoxy)ethyl) adipate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on reproductive organs were observed in the 90-day repeated dose toxicity study with the registration substance, which investigated additional fertility endpoints.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In the 90-day repeated dose toxicity study with the registration substance by the oral route and according to OECD 408, no adverse effects on the reproductive organs were observed (Charles River Laboratories, 2019).  No effect on the length and regularity of the estrous cycle, sperm parameters (motility, concentration, morphology), and weight and morphology of male and female reproductive organs were observed. Therefore, in accordance with Column 1 of REACH Annex IX, an extended one-generation reproductive toxicity study with the registration substance is not required.

Short description of key information:

No effects on reproductive organs were observed in the 90-day repeated dose toxicity study with the registration substance, which investigated additional fertility endpoints.

Effects on developmental toxicity

Description of key information

Developmental toxicity study (OECD 414) rat, oral:

NOAEL general toxicity and developmental toxicity in maternal animals >= 1000 mg/kg bw/day (highest dose tested)

NOAEL fetal developmental toxicty >= 1000 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April - 16 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 -14 weeks
- Weight at study initiation: approximately 185 to 297 g (females)
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. Except during designated procedures.
- Water: municipal tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: analysis were performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 40 - 68
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 July 2018 To: 19 July 2018

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 h after adding vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (1.125) and test item (1.1 at 25 °C). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Polyethylene glycol 400 (Merck, Darmstadt, Germany) was identified as suitable vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis in week 1 for homogeneity and concentration analysis. For homogeneity analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%. In addition, stability was tested in samples collected in week 1.
For concentration analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory.Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Stability analysis performed previously in the repeated dose 90-day oral toxicity study were considered (Study No. 20148924) for the develomental toxicity study. Stability of the test item in the vehicle was determined over 5 h at room temperature under normal laboratory light conditions (lowest and highest concentration). Duplicate sets of each sample (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ± 10% of the concentration determined by the initial analysis of each formulation.

Analysis showed that concentrations analyzed were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

days 6 to 20 post-coitum, inclusive

Frequency of treatment:

once daily

Duration of test:

until day 21 day post coitum

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a dose range finder study (Study No. 20148926). In this study 6 time-mated female Wistar Han rats per dose groups were administered the test substance at 300, 600 and 1000 mg/kg bw/day once daily oral gavage from day 6 to day 20 post-coitum, inclusive. No mortality occurred during the study period and treatment-related findings were noted in the high dose group only. The most sensitive was female no. 21 which had rales (up to a moderate degree) and a slightly shallow respiration on day 2 and days 9-11 (slight rales only), a hunched posture and piloerection (treatment Days 2-6), diarrhoea and/or slightly reduced faeces production (treatment Days 3-6), and a flat gait (treatment Days 3-6). Flat gait was also noted for another two high dose females (nos. 22 and 23) from treatment days 2-6. Salivation seen after dosing among animals of all test item treated groups on several days during the treatment period was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity. Incidental findings included general erythema (at a slight degree), alopecia and scales. As these findings occurred in control animals treated with the vehicle only, a relation to treatment with the test item could be excluded.
The slight body weight loss (2%) observed in the high dose group from days 6-9 post-coitum was mainly caused by female no. 21 which lost 12% of her weight in this period. An additional measurement performed on day 8 post-coitum indicated 15% body weight loss from days 6-8 post-coitum. In the subsequent treatment period, mean body weight gain recovered to control levels again. After correction for the gravid uterus mean body weight on day 21 post-coitum was lower in the high dose group as compared to the control group (2.6% versus 8.4%; not statistically significant). This was again caused largely by female no. 21 which had a body weight loss of 7.1% after correction for the gravid uterus weight. Body weight gain before and after correction for (gravid) uterus weight of low and mid dose group females was unaffected by the treatment.
Absolute and relative food consumption was reduced in the high dose group between days 6-9 postcoitum. Again this was largely caused by the markedly lower food consumption of female no. 21 which was about 70% lower as compared to group 4 average (absolute and relative to body weight). In the subsequent period, it recovered to normal values again. The statistically significantly higher mean value for relative food consumption observed in the mid dose group from post-coitum days 9-12 was not considered to be toxicologically relevant as changes were transient and mean value remained within the range of available historical control data.

Necropsy did not reveal any toxicologically relevant alterations. One low dose female was observed with enlarged parathymic lymphnodes. As this was an isolated finding at the low dose only, it was considered unrelated to treatment with the test item.

All females were pregnant, except for one female of the low dose. All pregnant females had viable fetuses, except for one control female which had resorptions only. A relation to treatment with the test item could be excluded, since females of the control group were treated with the vehicle alone.
There were no females that aborted or delivered before scheduled necropsy. The number of implantation sites, pre- and post-implantation loss, sex ratio and fetal body weights were unaffected by treatment. Mean numbers of corpora lutea were statistically significantly increased in the 300 and 1000 mg/kg groups, but not in the 600 mg/kg group. As treatment started after implantation of the conceptus, a relation to the test item could be excluded.

Litter sizes were within normal limits for all groups, the male:female ratios were equal in litters of all groups and fetal body weights were unaffected by treatment. The only external malformation noted was an inwards rotated right fore limb in a single control fetus (A003-01). As this malformation occurred in the control treated with the vehicle alone, a relation to treatment with the test-item could be excluded. No external variations were observed.

Based on the results of the dose range finder, selected dose levels for the main study were 300, 600 and 1000 mg/kg.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on day 2 post-coitum - up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (gavage study)

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: the number and distribution of live and dead fetuses, the number and distribution of embryo-fetal deaths, the sex of each fetus based on the ano-genital distance.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Other: As skeletal malformations were suspected for one animal of the low and one animal of the mid dose group,which were selected for visceral examination, these fetuses were also subjected to skeletal examination

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Descriptive statistics number, meanand standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss (%): ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / number of implantation sites) x 100
Number of viable fetuses affected/litter (%): (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: slight salivation was observed in high dose animals from day 4 of treatment onwards, and in a single female of the low dose group on the last day of treatment. Salivation was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). (non-adverse)

All other clinical signs noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be non-treatment-related.
(Please refer to table 1 in the "Any other information on results incl. tables" section.)

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: slightly (not statistically significantly) lower mean body weight gain after correction for gravid uterus was observed in high dose group females compared to concurrent control mean (19.0 vs 24.0 g) and in one female of the low dose group. The finding in the high dose group was mainly attributed to one female that appeared to have 0.3% body weight loss during the study period. In the absence of a dose-related trend, this observation was not considered toxicological relevant. (Please refer to table 2 and 3 in the "Any other information on results incl. tables" section.)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: absolute and relative food consumption between treatment days 6-9 was decreased by 16 and 12 %, respectively, when compared to controls, reaching statistical significance for relative values only. Food consumption, normalized over the next treatment days and remained within the normal range of biological variation over the rest of the treatment period and was therefore not considered toxicologically relevant (non-adverse). (Please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

control and 600 mg/kg bw/day: one female of each group had an early delivery on the morning of scheduled necropsy (day 21 post-coitum). In the absence of a dose-related trend this finding was considered to be unrelated to treatment.

Other incidental findings among control and treated animals included uterus with a wateryclear cyst (one control female) and alopecia. These findings are occasionally seen among rats used in these types of studies and therefore were considered changes of no toxicological significance. (Please refer to table 6 in the "Any other information on results incl. tables" section.)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

(Please refer to table 7 in the "Any other information on results incl. tables" section.)

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

control: relatively high mean values for post-implantation loss in the control group was caused by one female which had only one implantation that died early during gestation. Since this female was treated with the vehicle alone, a relation to the test item could be excluded. (Please refer to table 8 and 9 in the "Any other information on results incl. tables" section.)

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

control: relatively high mean values for total resorptions loss in the control group was caused by one female which had only one implantation that died early during gestation. Since this female was treated with the vehicle alone, a relation to the test item could be excluded. (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)

Early or late resorptions:

no effects observed

Description (incidence and severity):

control: one female had an early resorption (vehicle group, non-treatment related) (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)

Dead fetuses:

no effects observed

Description (incidence and severity):

(Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

control and 600 mg/kg bw/day: one female of each group delivered their offspring on the day of scheduled necropsy (day 21 post-coitum) and delivered 5 (control) and 8 (600 mg/kg bw/day) viable pups, all without developmental malformations. This early delivery was
considered non-treatment-related as it occurred at the same incidence in the 600 mg/kg group as in the vehicle control group. (Please refer to table 7 in the "Any other information on results incl. tables" section.)

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

control: one female was not pregnant (vehicle group, non-treatment related)
(Please refer to table 7 and 8 in the "Any other information on results incl. tables" section.)

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

general and developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed at this dose level

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the control, 300, 600 and 1000 mg/kg groups, respectively. These values remained well within the available historical control range. (Please refer to table 8 in the "Any other information on results incl. tables" section.)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

1000 mg/kg bw/day: one dead fetus observed (1/260 fetuses, incidental finding, non-treatment-related) (Please refer to table 7, 8 and 9 in the "Any other information on results incl. tables" section.)

Changes in sex ratio:

no effects observed

Description (incidence and severity):

(Please refer to table 8 and 9 in the "Any other information on results incl. tables" section.)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

control: slightly lower mean litter size in the control group was caused by one female which had one early resorption only. As this female was treated with the vehicle only, a relation to the test item could be excluded.
Mean litter sizes were 10.5, 11.7, 11.5 and 11.8 fetuses/litter for the control, 300, 600 and 1000 mg/kg groups, respectively. (Please refer to table 9 in the "Any other information on results incl. tables" section.)

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

300 and 600 mg/kg bw/day: one fetus of the low dose group had gastroschisis and missed one digit of a forepaw and one fetus of the mid dose group had a filamentous tail. (Additional skeletal examination revealed that the fetus of the low dose group appeared to have sternoschisis and vertebral anomaly with associated rib anomaly as well.) At the isolated incidence and in the absence of a dose-related trend, these malformations were considered to be of spontaneous origin. (Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: the externally malformed fetus of the low dose group also had vertebral anomaly and sternoschisis, skeletal examination revealed another vertebral anomaly in a littermate and bent limb bones (humerus and scapula) in a furtherlow dose fetus. At the isolated incidence and in the absence of a dose-related trend, these finidngs were considered to be of spontaneous origin, since all the malformations that were noted were observed previously in historical control fetuses.

Skeletal variations: Skeletal variations occurred at an incidence of 73.1%, 70.8%, 88.6% and 73.5% per litter in the control, 300, 600 and 1000 mg/kg groups, respectively.
600 mg/kg bw/day: statistically significantly higher incidence of total skeletal variations compared to the control. The reason for these increased incidences in this group is unknown, but as none single finding reached statistical significance or showed a dose relationship, they were not considered toxicologically relevant. Therefore, also the statistically significant higher incidence of total variations in this group was considered to be a chance finding.

All other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.
(Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral variations that were noted occurred in the absence of a dose-related incidence trend, occurred infrequently, and/or at frequencies that were within the range of available historical control data (non-treatment-related). (Please refer to table 10 and 11 in the "Any other information on results incl. tables" section.)

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: paw

external: tail

skeletal: sternum

skeletal: vertebra

other: External: gastroschisis

Description (incidence and severity):

At the isolated incidence and in the absence of a dose-related trend, these findings were considered to be of spontaneous origin, since all the malformations that were noted were observed previously in historical control fetuses.

Key result

Developmental effects observed:

no

Table 1: Summary of clinical signs

		Pre-treatment	Treatment
Dose group (mg/kg bw/day)	Week:	1xxx	1xxxxxxxxxxxxxx
Sign (Max grade, Location)	Day:	1234	123456712345671
Control
Gait / motility	G:	xxxx	xxx1xxxxxxxxxxx
Flat gait (1)	%:	xxxx	xxx0xxxxxxxxxxx
Skin / fur	G:	xxxx	xx1xxx111111111
Piloerection (1)	%:	xxxx	xx0xxx110000000
Fissures (3)	G:	xxxx	xxxx11111111111
(Flewes)	%:	xxxx	xxxx00000000000
300
Breathing	G:	xxxx	xxxxxxxxxxxxxx1
Rales (3)	%:	xxxx	xxxxxxxxxxxxxx0
Skin / fur	G:	xxxx	xx111xxxxxxxxxx
Piloerection (1)	%:	xxxx	xx000xxxxxxxxxx
Alopecia (3)	G:	xxxx	xxxxxxxxxxxxx11
	%:	xxxx	xxxxxxxxxxxxx00
Secretion / excretion	G:	xxxx	xxxxxxxxxxxxxx1
Salivation (3)	%:	xxxx	xxxxxxxxxxxxxx0
600
Skin / fur	G:	xxxx	xx1xx11111xxxxx
Piloerection (1)	%:	xxxx	xx0xx00000xxxxx
Secretion / excretion	G:	xxxx	xxxxxxxx1xxxxxx
Chromodacryorrhoea (3) (snout)	%:	xxxx	xxxxxxxx0xxxxxx
1000
Skin / fur	G:	xxxx	xxx111111111111
Piloerection (1)	%:	xxxx	xxx011122221000
Alopecia (3)	G:	xxxx	xx1111111111111
	%:	xxxx	xx0000000011111
Secretion / excretion	G:	xxxx	xxx111111111111
Salivation (3)	%:	xxxx	xxx379999AAAAAA

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%,..., A=more than 95%)

x: Observation performed, sign not present

 

Table 2: Summary of body weights (g)

Dose group (mg/kg bw/day)		Control	300	600	1000
Post Coitum Day 2	Mean	210	211	212	210
	ST.DEV.	22.5	20.1	15.2	16.3
	N	21	22	22	22
Day 6	Mean	224	225	225	226
	ST.DEV.	23.6	22.0	19.0	15.8
	N	21	22	22	22
Day 9	Mean	231	234	235	230
	ST.DEV.	24.5	22.6	17.9	16.6
	N	21	22	22	22
Day 12	Mean	246	249	250	245
	ST.DEV.	27.1	24.8	19.5	17.0
	N	21	22	22	22
Day 15	Mean	260	262	265	259
	ST.DEV.	29.1	25.8	20.4	19.2
	N	21	22	22	22
Day 18	Mean	288	293	297	289
	ST.DEV.	36.7	27.3	23.5	20.3
	N	21	22	22	22
Day 21	Mean	325	329	334	325
	ST.DEV.	44.4	30.8	24.6	22.9
	N	20	22	21	22

 

Table 3: Summary of body weight gain (%)

Dose group (mg/kg bw/day)		Control	300	600	1000
Post Coitum Day 2	Mean	-6	-6	-6	-7
	ST.DEV.	2.8	2.9	3.9	2.5
	N	21	22	22	22
Day 6	Mean	0	0	0	0
	ST.DEV.	0.0	0.0	0.0	0.0
	N	21	22	22	22
Day 9	Mean	3	4	4	2
	ST.DEV.	2.8	2.7	3.4	2.4
	N	21	22	22	22
Day 12	Mean	10	11	11	8
	ST.DEV.	2.9	3.1	3.6	2.6
	N	21	22	22	22
Day 15	Mean	16	16	18	15
	ST.DEV.	4.3	3.2	3.3	3.6
	N	21	22	22	22
Day 18	Mean	28	30	32	28
	ST.DEV.	7.9	4.5	3.9	4.2
	N	21	22	22	22
Day 21	Mean	44	46	48	44
	ST.DEV.	11.1	7.3	6.2	5.7
	N	20	22	21	22

 

 

Table 4: Summary of food consumption (g/animal/day)

Dose group (mg/kg bw/day)		Control	300	600	1000
Post coitum Days 2-6	Mean	22	22	22	23
	St.dev.	2.7	3.2	3.0	2.5
	N	21	22	22	22
Days 6-9	Mean	19	19	19	16
	St.dev.	2.8	3.7	3.3	4.4
	N	21	22	22	22
Days 9-12	Mean	21	22	21	20
	St.dev.	2.7	4.0	3.9	3.4
	N	21	22	22	22
Days 12-15	Mean	22	21	22	21
	St.dev.	3.6	3.5	3.3	2.6
	N	21	22	22	22
Days 15-18	Mean	23	23	24	22
	St.dev.	3.3	3.2	2.4	2.6
	N	21	22	22	22
Days 18-21	Mean	24	24	24	23
	St.dev.	3.9	3.5	2.6	2.9
	N	21	22	22	22
Mean of means		22	22	22	21

 

Table 5: Summary relative food consumption (g/kg body weight/day)

Dose group (mg/kg bw/day)		Control	300	600	1000
Post coitum Days 2-6	Mean	98	100	96	103
	ST.DEV.	9.6	9.6	10.9	10.4
	N	21	22	22	22
Days 6-9	Mean	80	81	83	70 *
	ST.DEV.	10.1	11.9	13.7	17.1
	N	21	22	22	22
Days 9-12	Mean	85	86	82	82
	ST.DEV.	6.8	9.4	13.9	11.8
	N	21	22	22	22
Days 12-15	Mean	85	82	84	82
	ST.DEV.	9.0	8.4	8.9	6.8
	N	21	22	22	22
Days 15-18	Mean	79	80	81	77
	ST.DEV.	4.3	6.2	4.6	6.5
	N	21	22	22	22
Days 18-21	Mean	75	72	74	71
	ST.DEV.	6.4	6.7	7.7	6.6
	N	21	22	22	22
Mean of means		84	83	83	81

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 6: Macroscopic findings

Dose group (mg/kg bw/day)	Control	300	600	1000
Post coitum Animals examined	22	22	22	22
Animals without findings	20	21	21	20
Animals affected	2	1	1	2
General observations Early delivery	1	0	1	0
Uterus Cyst(s)	1	0	0	0
Skin Alopecia	0	1	0	2

 

Table 7: Summary of maternal survival and pregancy status

Dose group (mg/kg bw/day)	control		300		600		1000
No.	%	No.	%	No.	%	No.	%
Females on study 22		22		22		22
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	0	0.0
Females that died	0	0.0	0	0.0	0	0.0	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females that were euthanized	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Females examined at scheduled necropsy	22#	100.0	22	100.0	22#	100.0	22	100.0
Nongravid	1	4.5	0	0.0	0	0.0	0	0.0
Gravid	21	95.5	22	100.0	22	100.0	22	100.0
With resorptions only	1	4.8	0	0.0	0	0.0	0	0.0
With viable fetuses	20	95.2	22	100.0	22	100.0	22	100.0

# Including females that delivered day 21 (control and 600 mg/kg bw/day dose group)

 

Table 8: Summary of fetal data at scheduled necropsy

		Sex		Viable Fetuses	Dead fetuses	Resorptions		Post-implantation		Corpora lutea	Pre-implantation loss	Fetal weight (g)	No. of gravid females
Dose group (mg/kg bw/day)		Male	Female			Early	Late	Loss	Sites
Vehicle	Total	119	101	220	0	17	0	17	237	239	2	NA	21
	Mean	5.7	4.8	10.5	0.0	0.8	0.0	0.8	11.3	11.4	0.1	5.3
	S.D.	2.15	2.06	3.43	0.00	1.21	0.00	1.21	3.13	3.14	0.30	0.40
300	Total	122	136	258	0	8	0	8	266	276	10	NA	22
	Mean	5.5	6.2	11.7	0.0	0.4	0.0	0.4	12.1	12.5	0.5	5.3
	S.D.	2.22	1.65	1.83	0.00	0.58	0.00	0.58	1.69	1.60	0.96	0.30
600	Total	127	125	252	0	5	0	5	257	263	6	NA	22
	Mean	5.8	5.7	11.5	0.0	0.2	0.0	0.2	11.7	12.0	0.3	5.2
	S.D.	1.57	1.73	1.60	0.00	0.53	0.00	0.53	1.64	1.56	0.55	0.24
1000	Total	124	135	259	1	15	0	16	275	280	5	NA	22
	Mean	5.6	6.1	11.8	0.0	0.7	0.0	0.7	12.5	12.7	0.2	5.1
	S.D.	2.22	2.19	1.48	0.21	0.84	0.00	0.94	1.30	1.28	0.43	0.40

None significantly different from control group

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea, fetal weights compared using dunnett's test

SD standard deviation

 

Table 9: Summary of fetal data at scheduled necropsy (% per litter)

	Dose group (mg/kg bw/day)	control	300	600	1000
Corpora Lutea	Mean	11.4	12.5	12.0	12.7
	S.D.	3.14	1.60	1.56	1.28
	N	21	22	22	22
Viable Fetuses (%)	Mean	88.4	96.9	98.2	94.2
	S.D.	23.53	4.86	4.18	7.16
	N	21	22	22	22
Dead Fetuses (%)	Mean	0.0	0.0	0.0	0.4
	S.D.	0.00	0.00	0.00	1.77
	N	21	22	22	22
Early Resorptions (%)	Mean	11.6	3.1	1.8	5.4
	S.D.	23.53	4.86	4.18	6.27
	N	21	22	22	22
Late Resorptions (%)	Mean	0.0	0.0	0.0	0.0
	S.D.	0.00	0.00	0.00	0.00
	N	21	22	22	22
Total Resorptions (%)	Mean	11.6	3.1	1.8	5.4
	S.D.	23.53	4.86	4.18	6.27
	N	21	22	22	22
Pre-Implantation Loss (%)	Mean	0.8	3.5	2.3	1.8
	S.D.	2.50	7.54	4.60	3.34
	N	21	22	22	22
Post-Implantation Loss (%)	Mean	11.6	3.1	1.8	5.8
	S.D.	23.53	4.86	4.18	7.16
	N	21	22	22	22
Males (%)	Mean	54.7	46.4	50.6	47.7
	S.D.	12.55	15.90	12.26	17.26
	N	20	22	22	22
Females (%)	Mean	45.3	53.6	49.4	52.3
	S.D.	12.55	15.90	12.26	17.26
	N	20	22	22	22
Male fetal weights (g)	Mean	5.5	5.4	5.4	5.2
	S.D.	0.40	0.35	0.30	0.36
	N	20	22	22	22
Female fetal weights (g)	Mean	5.2	5.2	5.1	5.0
	S.D.	0.45	0.30	0.20	0.44
	N	20	22	22	22
Combined fetal weights (g)	Mean	5.3	5.3	5.2	5.1
	S.D.	0.40	0.30	0.24	0.40
	N	20	22	22	22

proportional (%) data compared using the mann-whitney test

corpora lutea and implantation sites compared using dunnett's test

fetal weights compared using dunnett's test

none significantly different from control group

SD standard deviation

N number

 

Table 10: Summary of fetuses and litters with malformation (absolute number)

	Fetuses				Litters
Dose group (mg/kg bw/day)	control	300	600	1000	control	300	600	1000
Number examined externally	220	258	252	259	20	22	22	22
Trunk- gastroschisis	0	1	0	0	0	1	0	0
Tail- absent, short or filamentous	0	0	1	0	0	0	1	0
Ectrodactyly	0	1	0	0	0	1	0	0
Number examined viscerally	110	130	128	129	20	22	22	22
Number with findings	0	0	0	0	0	0	0	0
Number examined skeletally	110	129	125	130	20	22	22	22
Vertebral anomaly with or without associated rib anomaly	0	2	0	0	0	1	0	0
Bent limb bone(s)	0	1	0	0	0	1	0	0
Sternoschisis	0	1	0	0	0	1	0	0
Total number with malformations
External :	0	1	1	0	0	1	1	0
Soft tissue :	0	0	0	0	0	0	0	0
Skeletal :	0	3	0	0	0	2	0	0
Combined :	0	3	1	0	0	2	1	0

 

Table 11: Summary of fetuses and litters with variations (absolute number)

	Fetuses				Litters
Dose group (mg/kg bw/day)	control	300	600	1000	control	300	600	1000
Number examined externally	220	258	252	259	20	22	22	22
Number with findings	0	0	0	0	0	0	0	0
Number examined viscerally	110	130	128	129	20	22	22	22
Liver- small supernumerary lobe(s)	4	1	1	6	3	1	1	6
Ureter(s)- convoluted	0	3	1	0	0	3	1	0
Ureter(s)- dilated	0	1	0	0	0	1	0	0
Renal papilla(e)- absent and/or small	0	1	0	0	0	1	0	0
Number examined skeletally	110	129	125	130	20	22	22	22
14th rudimentary rib(s)	59	74	78	71	18	22	21	19
14th full rib(s)	7	7	20	6	6	5	11	4
Bent rib(s)	12	12	24	14	7	7	14	7
Pelvic girdle- caudal shift	5	9	13	4	5	6	8	3
Reduced ossification of the skull	9	8	16	10	7	6	10	5
Sternebra(e) malaligned	9	14	13	10	8	12	11	6
Scapula(e)- bent	0	0	2	0	0	0	2	0
Metacarpal(s) and/or metatarsal(s) unossified	1	1	0	7	1	1	0	5
7th cervical ossification site(s)	4	7	8	6	4	7	7	4
Sternebra(e)- branched	2	0	1	1	2	0	1	1
Vertebral centra- reduced ossification	0	1	0	1	0	1	0	1
7th cervical full rib(s)	0	2	0	0	0	2	0	0

Effect on developmental toxicity: 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:

The available information comprises adequate and reliable studies (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.7, of Regulation (EC) No 1907/2006.

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

- Developmental toxicity/teratogenicity

Developmental toxicity was tested in a GLP compliant study in Wistar rats (Crl: WI(Han)) according to OECD 414 (Charles River Laboratories, 2019). In this study 22 timed-mated rats per dose level were treated daily by gavage with 300, 600 and 1000 mg test substance /kg bw/day suspended in polyethylene glycol 400 from gestation day 6 to day 20. The animals of the control group received vehicle only (polyethylene glycol 400).

 

Analysis of test substance formulations showed that analyzed concentrations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).

 

Examinations of maternal animals included clinical observations, mortality, body weight, food consumption gross pathological examinations and abortions or delivery before scheduled necropsy. At day 21 of gestation fetuses were delivered by caesarian section. The following parameters were determined at caesarian section: number of corpora lutea, number of implantations, implantation loss, uterus weight, number of resorptions (early, late and total), number of pregnant, number of live/dead fetuses, sex of live fetuses, individual weights of fetuses/litter and external (all fetuses), visceral (one half of the fetuses) and skeletal (one half fetuses and two additional fetuses with suspicious findings during visceral examination) malformations and variations.

There were no test substance-related deaths.

 

No treatment-related toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, body weight, food consumption and macroscopic examination) in the 300, 600 and 1000 mg/kg groups. The number of pregnant, the mean number of corpora lutea, implantation sites, resorptions (total, early and late), live/dead fetuses were comparable across all treatment groups.

 

No treatment-related effects were noted on any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations) in the 300, 600 and 1000 mg/kg bw/day groups.

Fetal examinations did not reveal visceral malformations and visceral variations that were noted, occurred in the absence of a dose-related incidence trend, occurred infrequently, and/or at frequencies that were within the range of available historical control data and were thus considered non-treatment-related. Single incidences of external and skeletal malformations were observed, but considered incidental findings that were not-related to the test substance. One fetus of the low dose group had gastroschisis and missed one digit of a forepaw and one fetus of the mid dose group had a filamentous tail. The externally malformed fetus of the low dose group also had vertebral anomaly and sternoschisis, skeletal examination revealed another vertebral anomaly in a littermate and bent limb bones (humerus and scapula) in a further low dose fetus. At the isolated incidence and in the absence of a dose-related trend, these findings were considered to be of spontaneous origin, since all the malformations that were noted were observed previously in historical control fetuses. Skeletal variations occurred at an incidence of 73.1%, 70.8%, 88.6% and 73.5% per litter in the control, 300, 600 and 1000 mg/kg groups, respectively. In the mid dose group a statistically significantly higher incidence of total skeletal variations compared to the control was observed. The reason for these increased incidences in this group was unknown, but as none single finding reached statistical significance or showed a dose relationship, they were not considered toxicologically relevant. Therefore, also the statistically significant higher incidence of total variations in this group was considered to be a chance finding. All other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.

Based on the above reported findings a no-observed-adverse-effect level (NOAEL) for maternal (general and developmental) toxicity and fetal developmental toxicity was determined at 1000 mg/kg bw/day, the highest dose level tested.

Justification for classification or non-classification

The available data on reproductive and developmental toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.