2,2'-p-phenylenedioxydiethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was undertaken with regard to concentration and homogeniety on Weeks 1,3 and 6 of the study.

Duration of treatment / exposure:

The males were treated for 2 weeks prior to mating, through mating and until necropsy after 4 weeks of treatment.
The females were treated for 2 weeks prior to mating, throughout mating, gestation and until termination on ca. Day 4 of lactation.

Frequency of treatment:

Once daily, 7 days per week

Remarks:

Doses / Concentrations:
100 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Daily, commencing one week prior to treatment until termination

FOOD CONSUMPTION: Yes
- Time schedule: Weekly

WATER CONSUMPTION: Yes
- Time schedule: Weekly

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: the number of live pups and number found dead were recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the precense of milk in the stomach and for any externally visible abnormalities daily until Day 4 of lactation.

Postmortem examinations (parental animals):

GROSS NECROPSY: the cranial, thoracic and abdominal contents were examined macroscopically

ORGAN WEIGHTS: For males the testes and epididymides.

HISTOPATHOLOGY: For males each of the testes and epididymides. For females each of the ovaries.
Both for top dose and control animals only.

Statistics:

Body weight and food comsumption data were subjected to analysis of variance or the Kruskal-Wallis non-parametric analysis as appropriate.
Organ weight data were analysed by analysis of variance and analysis of covariance.
Histology data were analysed by Fischers Exact Probability test

Reproductive indices:

The following fertility indices were evaluated for each group: Fertility index (male), Fertility index (female), Gestation Index.

Offspring viability indices:

The following fertility indices were evaluated for each group: Birth Index, Live Birth Index, Viability Index

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of treatment-related effects

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of treatment-related effects

Reproductive effects observed:

not specified

Conclusions:

There were no obvious effects on parental toxicity, nor were there any obvious effects of treatment on fertility, pregnancy performance or pup development. The NOEL was 1000 mg/kg for both adults and pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to internationaly accepted guideline and GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

Justification for selection of Effect on fertility via oral route: Only one study available. The study is Klimisch 1.

Additional information

In an OECD 421 reproductive toxicity screening study there were no obvious effects on parental toxicity, nor were there any obvious effects of treatment on fertility, pregnancy performance or pup development. The NOEL was 1000 mg/kg for both adults and pups.

Effects on developmental toxicity

Description of key information

The substance does not have developmental toxicity effects after oral repeated dose exposure of pregnant rats (14 -days)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29/12/2015-29/11/2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test item information for group 1-4:
Used between 21 February 2016 and 9 March 2016 for Group 1-4
- Identification 2,2'-p-phenylenedioxydiethanol HQEE)
- Appearance: white solid
- Batch: 15DR198795
- Purity/Composition: 99.8%
- Test item storage: room temperature
- Expiry date: 27 July 2016


Test item information for group 5-6:
Used between 4 October 2016 and 19 October 2016 for group 5-6
- Identification 2,2'-p-phenylenedioxydiethanol HQEE)
- Appearance: white solid
- Batch : 16DR070651
- Purity/composition; 99.4%
- Expiry date: 24 August 2016


Test item information for group 7-8:
Used between 06 November 2016 and 21 November 2016 for Group 7-8
- Identification 2,2'-p-phenylenedioxydiethanol HQEE)
- Appearance: white solid
- Batch: 16DR205726
- Purity/composition: 99.7%
- Expiry date: 24 October 2017

Material Number: 101376
Molecular formula: C10H14O4
Molecular weight: 198.2158
Solubility in water: 13.1 g/L at 21°C
Stability in water: Chemical analyses of formulation were performed to assess accuracy, homogeneity and stability over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator in the dark.

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number of animals: F0-generation: 176 females / F1-generation: 1398 fetuses
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: approximately 220 g on average basis
- Housing: Females were individually housed in Macrolo plastic cages. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum):Free access to tap-water
- Acclimation period: at least 5 days prior treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (range of actual daily mean: 21.1-22.3°C)
- Humidity (%): relative humidity of 40 to 70% (range of actual daily mean: 37.83-60.12%)
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study

Diet, water, bedding and cage-enrichment/nesting material evaluation for containments and/or nutriments was performed according to facility standard procedures. There were no findings that could interfere with the study.

IN-LIFE DATES:
Group 1-4 From 16-18 February 2016 To: 07-10 March 2016
Group 5-6 From 29 September 2016 To: 19-20 October 2016
Groupe 7-8 From 01 November 2016 To 21-22 November 2016

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Method formulation:
- For group 1-6, formulation (w/w) were prepared within 8 days prior to dosing.
- For group 7-8, formulation (w/w) were prepared daily within 5 hours prior to dosing.
All formulations were homogenized to a visually acceptable level. No adjustement was made for specific gravity/densitiy of the test item. No correction was made for the purity/composition of the test item.
Formulations were placed on a magnetic stirrer during dosing.


FORMULATINON ANALYSIS:
Formulation analysis was performed. No test item was detected in the Group 1 and Group 7 formulations (all control groups).
The concentration analysed in the formulations of Group 2, 3, 4 and 8 were agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).
The formulations of Group 2, 4 and 8 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.


VEHICLE
- Concentration in vehicle: Group 1: 0mg/kg bw; Group 2: 100 mg/kg bw; Group 3: 300 mg/kg bw; Group 4: 1000 mg/kg bw: Group 5: 0 mg/kg bw (Coutrol for Group 6); Group 6: 2000 mg/kg bw; Group 7: 0mg/kg bw (Control for Group 8); Group 8: 2000 mg/kg bw
- Amount of vehicle (if gavage): Group 1-4: 5 mL/kg bw; Group 5-8: 10 mL/ kg bw (actual dose volume were calculated according to the lastest body weight)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations for groups were taken during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Sample of formulation were taken for accuracy (all concentration) and homogeneity (Group 2, 4, 6 and 8) of the preparation.

Details on mating procedure:

Untreated females were mated at the Supplier, and were delivered at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive (14 days)

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

21 days

Dose / conc.:

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

Remarks:

Group1: Control group for Group 2, 3, 4

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

Dose / conc.:

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

Remarks:

Group 5: Control group for Group 6

Dose / conc.:

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

Remarks:

Group 6: Due to a formulation error, the inteded dose level in Group 6 (2000 mg/kg bw) was not reached.
The concentration of the formulation for Group 6 was approximately 2 times lower than intended and the animals were dosed at approximately 970 mg/kg instead of the inteded dose level of 2000 mg/kg bw.

Dose / conc.:

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

Remarks:

Group 7: Control group for Group 8

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Group 8

No. of animals per sex per dose:

22 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on results of the 14-Day Dose Range Finding study. The dose level for Group 6 was selected based on the results in Group 1-4.
The dose level for Group 8 was the same as the intended dose level in Group 6, since this dose level was not reached in Group 6.
Due to a formulation error, the inteded dose level in Group 6 was not reached during Day 9-15 or 8-14 post-coitum, depending on mating date.
The concentration of the formulation for Group 6 was approximately 2 times lower than intended and the animals were dosed at approximately 970 mg/kg instead of the inteded dose level of 2000 mg/kg bw.

- Rationale for animal assignment:
Randominization: on the day after receipt, by computer-generated random algorithm according to body weight, with all animals within +/-25% of the mean per subgroup. Females which were mated o the same day are classified in the same subgroup.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from Day 2 post-coitum onwards up to the day prior the necropsy.
Clinical observations were conducted at least immediately after dosing.

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

FOOD CONSUMPTION: Yes
- Time schedule for examination: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION :Yes
- Time schedule for examinations: subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
All animals were sacrified on Day 21 post-coitum using oxygen/carbon dioxide procedure and subsequently subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.
All macroscopic abnormalities were recorded and collected (fixed in 10% buffered formalin).

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
- Number and distribution of live and dead fetuses: Yes
- Weight of each fetuses: Yes
- Sex of each fetuses from the ano-genital fistance (during necropsy) and also from gonadal inspections (during further fetal examination): Yes
- Externally visible macroscopic fetal abnormalities: Yes

In cased implatations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites.

Fetal examinations:

- External examinations: Yes: each viable fetus was examined in detail, weighted and sexed
- Soft tissue examinations: Yes: Approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. Thoracic and abdominal cavities were opened and the heart and major vessels were examined. Fetal kidneys were examined and graded for renal papillae development.
Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded.
- The sex of all fetuses was confirmed by internal examination.
- Skeletal examinations: Yes: from the other one-half of the fetuses (live and dead) in each litter (all groups), the sex was confirmed by internal examination. No skeletal examination was performed for the fetuses of Groups 5 and Groups 6 after fixation in ethanol tissues were discarded.
- Head examinations: Yes: the heads were removed from theses fetuse for soft-tissues examinations of group 1-4 and 7-8. After examination, the tissues were discarded. No soft tissue examination of heads were performed for the fetuses of Groups 5 and 6, tissues were discarded.

Statistics:

The followins statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution
- The Fisher Exact -test was applied to frequency data
- The Mann Whitney test was used to compare mean litter proportions(% of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre-and post-implantations loss, and sex distribution.
- Mean litter proportions (% 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. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn's test was used to compare the compound-treated groups to the control group.

Group 2-4 and Group 8 were compared to their concurrent controls Group 1 and Group 7

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

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signes were noted up to 2000 mg/kg bw.
One female treated at 300 mg/kg showed restless behavior for 5 days during treatment, which was considered to be incidental and not related to treatment. The incidence and scabs and alopecia remained with the range of background findings for rats of this strain and age.

Mortality:

no mortality observed

Description (incidence):

No mortality occured during this study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight, body weight gain and body weight corrected for weight of the uterus were not adversely affected by treatment up to 2000 mg/kg bw.
At Day 15 post-coitum body weight gain in 2000 mg/kg bw treated females was slightly reduced compared the concurrent control females. As the effect was minor and recovered during subsequent days, it was not considered adverse in nature.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on food consumption (absolute and relative) up to 2000 mg/kg bw.
A slight but significant reduction in absolute (5-9% decrease versus control) and relative food consumption was noted between Day 6 and Day 15 post-coitum in females treated at 2000 mg/kg bw (Group 8) compared to their concurrent controls (Group 7).
However, as the effect was only slight, all values within normal limits, and no adverse concurrent effect on body weight was observed, this was considered not adverse but dose and test item related.

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:

no effects observed

Description (incidence and severity):

No treatment related macroscopic findings were observed up to 2000 mg/kg.
The incidence of incidental findings amoung control and treated animals was within the background range of findings that are encountered among rats of this age and this strain, and did not show a dose-related incidence trend.
These necropsy findings were therefore considered to be of no toxicological relevance.

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

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no treatment related effect on litter size for any group.
Mean litter sizes were 10.4, 10.9, 11.2 and 10.0 for the first control, 100, 300 and 1000 mg/kg bw groups, respectively and 10.3 and 11.2 for te second control and 2000 mg/kg bw group.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The number of early resorption at 1000 mg/kg was increased compared to the concurrent vehicle control group (10.4% per litter vs 2.8% per litter, respectively).
An extra dose group of 2000 mg/kg bw was added to this study.
At 2000 mg/kg bw the incidence of early resorption was not increased compared to the concurrent control (4.0% per litter compared to 6.6% per litter, respectively).
As there was no dose response relationship, the increased incidence of early resorption at 1000 mg/kg bw was considered incidental and not related to treatment with the test item.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One female treated at 100 mg/kg was not pregnant. All other females were pregnant with viable fetuses.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

maternal abnormalities

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no treatment related effect on mean fetal body weight up to 2000 mg/kg bw.
Mean combined (male and female) fetal body weights were 5.3 grams for the first control group up to 1000 mg/kg bw.
At 2000 mg/kg bw, mean combined fetal body weight were significantly lower compared the concurrent control group (i.e. 5.5 and 5.2 grams, respectively).
However, the values of the vehicle control group were above historitical control data, whereas the values for the 2000 mg/kg bw group were within these data
Therefore, the slightly reduced mean combined fetal weights at 2000 mg/kg bw were not considered toxicologically relevant.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 2000 mg/kg bw.
The significant differences in male:female ratio at 2000 mg/kg bw can be explained by the slightly skewed distribution towards male fetuses in the concurrent control group.
As the latter group was treated with the vehicle only, this cannot be contributed to treatment with the test item.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment related effect on litter size for any group.
Mean litter sizes were 10.4, 10.9, 11.2 and 10.0 for the first control, 100, 300 and 1000 mg/kg bw groups, respectively and 10.3 and 11.2 for te second control and 2000 mg/kg bw group.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no external developmental malformations or variations up to 2000 mg/kg bw.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to 2000 mg/kg bw.

There were two skeletal malformations observed, bent limb bones and vertebral anomaly without rib anomly.
The first malformation (bent limb bones) was in found fetuses of control.
The second one (vertebral anomaly without rib anomaly) was obsered in one fetus at 2000 mg/kg bw. The variations that were noted occured in the absence of a dose-related incidence trend, occured infrequently or stay within the ranges of the historitical data.
Therefore, all skeletal findings were not considered to be treatement related.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment effects on visceral morphology following treatment up to 2000 mg/kg bw.

Three fetuses, one in the control, 100 and 1000 mg/kg groups each, had situs inversus with all organs laterally transposed.
In two of these fetuses, the situ inversus was observed together with presence of one lung lobe at both sides and the latter also had a misshapen heart.
Because all malformations occured singly and/or in a control fetus, these were considered to be chance findings and not attributed to treatment.

Description (incidence and severity):

The number of fetuses (litters) initially available for morphological examination were 228, 229, 247 and 221 in Groups 1, 2, 3 and 4, respectively.
To further investigate the increased number of early resorption that occured at 1000 mg/kg bw, an additional control and 2000 mg/kg bw group were added. These respective additional groups resulted in 227 and 246 extra fetuses.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to 2000 mg/kg bw

Abnormalities:

no effects observed

Developmental effects observed:

no

Formulation Analysis

No test item was detected in the Group 1, Group 5 and Group 7 formulations (all control groups). The concentrations analysed in the formulations of Group 2, 3, 4, 6 and 8 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

The formulations of Group 2, 4, 6 and Group 8 prepared were homogeneous (i.e. coefficient of variation ≤ 10%). The long term storage samples were stable at ≤-70°C for 126 days.

Summary of Dose Range Finding Study:

In order to set the dose levels for the main teratology study, a dose range finding study was conducted.

Four groups of 6 females were expossed to 0, 100, 300 and 1000 mg/kg bw for Days 6 to 20 post-coitum inclusive by oral gavage.

Results:

• No mortality occured during the study period.
• No toxicologically significant clinical signs were noted up to 1000 mg/kg. Scabs noted for female n° 4 (control group) were within the range of backgroup findings for rats of this strain and this age.
• Body weight, body weight gain and body weight gain corrected for weight of the uterus were unaffected by the treatment up to 1000 mg/kg bw.
• Overall, food consumption (relative and absolute) remained within the same range os the vehicle control animals. Between Day 12 -15 post-coitum, relative food consumption was slightly, but significantly reduced in the 1000 mg/kg bw treatment group. As the reduction was only minor and without concurrent effect on body weight, this was considered of no toxicological relevance.
• No macroscopic findings were observed at necropsy in any of the groups.
• All females were pregnant with viable fetuses. No treatment related changes for the number of post-implantation loss were observed up to 1000 mg/kg bw.

Fetal findings:

• Litter sizes were within normal limits for all groups.
• The male:female ratios were equal in litters of all groups.
• Fetal body weight were unaffected by treatment up to 1000 mg/kg bw.
• External examination of the fetuses did not show any treatment related abnormalities.
• For one fetus at 1000 mg/kg, subcutaneous edema was observed. Visceral screening showed cranioschisis and thoracoschisis, which was confirmed by skeletal evaluation. Furthermore, a vertebral anomaly, general reduced ossification, unossified sternebrae n°5 and n°6, unossified metacarpals/metatarsals and 14th full ribs were observed. These findings were considered to be incidental and not related to treatment.

Conclusion:

Based on the results of the dose range finding study, selected dose for the main study were 100, 300, 1000 mg/kg bw.

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Elevel (NOAEL) for the test substance was established as being at least 2000 mg/kg.
The developmental NOAEL was established as being at least 2000 mg/kg.

Executive summary:

The Prenatal developmental toxicity study of the test substance in rats by oral gavage was performed according to the OECD 414 guideline and under GLP.

In the first part of the study, 88 mated females Wistar Han rats were assigned to 4 groups. The test item was administered once daily by oral gavage from Day 6 to 20 post-coitum at dose of 100, 300 or 1000 mg/kg (Group 2, 3 and 4 respectively). The animals were treated once daily for 7 day per week at volume of 5 mL/kg. Based on the preliminary results of the group 4 (1000 mg/kg), a possible treatment related increase in the incidence of early resorptions at the high dose was suspected. To investigate this further, it was deemed necessary to add an extra dose group. Since there was no maternal toxicity observed up to 1000 mg/kg, it was considered feasible to test a dose level of 2000 mg/kg (in a volume of 10mL/kg)

For the second part of the study, 44 mated females were assigned to 2 groups (Groups 5 and 6) of 22 females each. The test substance was administered once daily by oral gavage from Day 6 to 20 post-coitum.

However, due to a formulation error, dose levels in Group 6 were approximately 970 mg/kg instead of 2000 mg/kg during Day 9 -15 or 8 -14 post-coitum, depending on mating date. The concentrations of the formulations and consequently the dose levels for Group 6 were approximately 2 times lower than intended (970 mg/kg instead of 2000 mg/kg). Therefore, the animal data recorded for this group and its concurrent control were not included in the toxicological evaluation and no skeletal examinations was performed.

For the third part of the study, additional 44 animals were allocated into 2 groups to received either the vehicle, water, only (Group 7) or the test substance at 2000 mg/kg (Group 8), once daily by oral gavage from Day 6 to 20 post-coitum.

For all the groups, the females were checked daily for the presence of clincal signs. Food consuption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analysed for accuracy and homogeneity.

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded.

Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All lived fetuses were euthanized. One half of the fetuses were decapitated, these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained for skeletal examinations.

The results of the treatment of the mated females did not show any maternal toxicty in the 100, 300, 1000 and 2000 mg/kg groups. A (non-adverse) statistically significant decrease in food consumption was observed in dams dosed at 2000 mg/kg between Day 6 to 15 post-coitum.

Moreover, no developmental toxicity was observed up to 2000 mg/kg.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test substance was established as being at least 2000 mg/kg/day.

The developmental NOAEL was established as being at least 2000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted according to internationally accepted guideline.

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

For this endpoint, there is one OECD 414 study available assessing toxic effects of the test item after oral dosing. No studies are available for inhalation or dermal route. However, the data available is sufficient for classification purposes and the oral route is the most relevant route of administration for exposure in human.

The study (K.CG. Hartman - Van Dycke, 2017) is a Prenatal Developmental Toxicity study in rats, performed to generate information concerning the effects of the test item on females reproductive performance , on developmental effects in pups (malformations) and possible health effects hazards likely to arise from repeated exposure over a relatively limited period of time. This study was according to OECD Guideline 414.

The test item was administered once daily by oral gavage from Day 6 to 20 post-coitum at dose of 100, 300 or 1000 mg/kg (Group 2, 3 and 4 respectively) to 88 mated females Wistar Han (22 females per group). The rats of the Group 1 received the vehicle, water, alone.

The animals were treated once daily for 7 day per week at volume of 5 mL/kg (Group 1 -4). Based on the preliminary results of the group 4 (1000 mg/kg), a possible treatment related increase in the incidence of early resorptions at the high dose was suspencted. To investigate this further, it was deemed necessary to add an extra dose group. Since there was no maternal toxicity observed up to 1000 mg/kg, it was considered feasible to test a dose level of 2000 mg/kg.

Then, additional 44 mated females were assigned to 2 additional groups (Groups 7 and 8 each received of 22 females). The test substance was administered once daily by oral gavage from Day 6 to 20 post-coitum at a dose of 2000 mg/kg.

For all the groups, the females were checked daily for the presence of clincal signs. Food consumption and body weight were determined at periodic intervals.

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Gross lesions were collected and fixed from all animals at necropsy. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded.

Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All lived fetuses were euthanized. Half of the fetuses were decapitated, these fetuses were dissected and examined for visceral anomalies. The other half of the fetuses were processed and stained for skeletal examinations.

The results of the treatment of the mated females did not show any maternal toxicty in the 100, 300, 1000 and 2000 mg/kg groups. A (non-adverse) statistically significant decrease in food consumption was observed in dams dosed at 2000 mg/kg between Day 6 to 15 post-coitum.

Moreover, no developmental toxicity was observed up to 2000 mg/kg.

The developmental NOAEL was established as being at least 2000 mg/kg/day.

Justification for classification or non-classification

The substance did not induce any adverse effects in a Reproductive/Developmental Toxicity Screening Test and a Prenatal Developmental Toxicity Test in rats.

Therefore, according to the criteria set out in the CLP Regulation No 1272/2008 in section 3.7, the substance should not be classified as toxic for the reproduction.