Acetic acid, 2-chloro-, reaction products with 2-C11-13-alkyl-4,5-dihydro-1H-imidazole-1-ethanol and sodium hydroxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 February 2022 - 15 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C13 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid

EC Number:

938-645-3

Cas Number:

1689515-39-6

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C13 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid

Test material form:

liquid

Details on test material:

- Physical appearance: clear yellow liquid
- Storage conditions: At room temperature

Specific details on test material used for the study:

- Physical appearance: clear yellow liquid
- Storage conditions: At room temperature
- Test item handling: No specific handling conditions required

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 188-256 g
- Housing: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material, animals were housed individually. For psychological/environmental enrichment and nesting material, animals were provided with paper and with aspen wooden sticks, except when interrupted by study procedures/activities.
- Diet: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany (pellets)
- Water: ad libitum, municipal tap water
- Acclimation period: at least 5 days prior to dosing

Results of analysis of food and water for contaminants are provided by the Supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20 (actual range)
- Humidity (%): 49-51 (actual range)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 February 2022 To: 25 February 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
At least weekly new test item formulations were prepared stored at 4°C. Dose formulation were divided into aliquots where required and kept at room temperature until dosing. An adjustment was made for the specific gravity of the test item. A factor 2 was used to correct for the composition of the test item. The dose volume for each animal was based on the most recent body weight measurement. The dose formulations were continuously stirred during dosing.

VEHICLE
- Concentration in vehicle: 59, 177 and 590 mg/mL
- Amount of vehicle (if gavage): 1.695 mL/kg bw

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 of treatment for group 1, 2 and 3 to test for concentration and at group 2 and 3 to test for homogeneity.
The acceptance criteria for concentration was that the mean sample concentration results were within or equal to ± 10% of theoretical concentration.
Analyses were performed using a validated analytical procedure (test facility study 20297952).
For homogeneity the acceptance criteria was that the relative standard deviation of concentrations was ≤10% for each group.
Stability analyses was performed previously in conjunction with the method development and validation study of the test laboratory (test facility study 20297952). It demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

Once daily

Duration of test:

Day 6 to 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on of a Dose Range Finder study (test facility study 20297966) with oral exposure of the test material in rats with the same laboratory, and in an attempt to produce graded responses to the test item. In this study, dosing with 1000 mg/kg bw/day resulted in lower food consumption. No other maternal of developmental toxicity was observed. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no fasting
- Time of day for (rat) dam blood sampling: Day 21 post-coitum

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily beginning upon arrival through termination/release a check for mortality was done. At least once daily 0 to 1 hours post-dose, staining on Day 6 post coitum up to and including the day prior to necropsy case side observations were performed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15, and 21 post-coitum

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

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: regular basis throughout the study by visual inspection of the water bottles (data not reported)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-coitum day 21 euthanized by carbon dioxide inhalation
- All animals were subjected to an external, thoracic and abdominal examination, with special attention paid to the reproductive organs.
- Thyroid gland was weighed at necropsy, except for females that delivered their offspring prior to necropsy.
- Organs weights were recorded for all animals at necropsy. They were not recorded for animals found dead, euthanized in poor condition or in extremis or that delivered their off spring prior to necropsy.
- Thyroid gland and macroscopic abnormalities were collected, fixed and evaluated microscopically.

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

In case no macroscopically visible implantation sites are present, nongravid uteri will be stained using the Salewski technique in order to detect any former implantation sites.

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: 1.0 mL, sampled between 07.00 and 09.00 from the jugular vein
- Fasting: No
- Thyroid hormone parameters: TSH, T3 and T4

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
- Anogenital distance of all live rodent pups: Yes
- Other: body weight, sex ratio, litter size

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% and 5% levels.
Analyses were performed according to the statistical matrix presented in "Any other information on materials and methods incl. tables"
- Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.
- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall
Kruskal-Wallis test was found to be significant, then the above pairwise comparisons was
conducted using Dunn’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

Pregnancy rate (%) = (No. of pregnant females / No. of mated females) x 100
Male fetuses (%) = (No. of male fetuses / No. of fetuses) x 100
Female fetuses (%) = (No. of female fetuses / No. fetuses) x 100
Pre-Implantation Loss (%) = ((No. of corpora lutea - No. of implantation) / No. corpora lutea) x 100
Post-Implantation Loss (%) = ((No. of implantations - No. of live fetuses ) / No. implantations) x 100
Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding / No. of fetuses in litter examined) x 100
Calculated intervals for BW gain: Days 2 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, and 6 to 21 post-coitum
Gravid uterus adjusted BW: BW on Day 21 post-coitum - BW on Day 6 post-coitum - gravid uterus weight

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal breathing sounds were observed for 1/22, 4/21 and 2/22 surviving females at 100, 300 and 1000 mg/kg bw/day, respectively, on one or several days during the Treatment Period. Salivation was seen after dosing in 1/22 females at 100 mg/kg bw/day and in all females at 1000 mg/kg bw/day during the Treatment Period. Taking into account the nature and minor severity of the effect and its time of occurrence (i.e., directly after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity. Any other clinical signs noted during the Treatment Period 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 unrelated to treatment with the test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test material-related mortality was observed.
One female in the 300 mg/kg bw/day group was sacrificed in extremis on Day 9 post-coitum, as it was noted with abnormal breathing sounds, hunched posture, erected fur and a swollen abdomen on this day. In addition, food consumption was reduced over Days 6-9 post-coitum, correlating to 11% body weight loss over this period. At necropsy, the small and large intestine (i.e., duodenum, ileum, jejunum, cecum and colon) were filled with gas. This was the only animal in whole study that did not survive until scheduled necropsy and no severe body weight loss, reduced food consumption, or findings in the intestines were observed in any other animal in the study, including the highest dose group of 1000 mg/kg/day. Based on these observations, the death of this animal was considered not to be related to treatment with the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights, body weight gain and gravid uterus adjusted body weight gain of test material-treated animals remained in the same range as control over the Treatment Period. The statistically significantly higher body weight gain for females at 1000 mg/kg bw/day over Days 9-12 post-coitum was considered unrelated to treatment with the test material as it concerned an increase and no trend regarding duration of treatment was apparent.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test material-related changes in food consumption were noted.

Endocrine findings:

no effects observed

Description (incidence and severity):

Note: Lower limits of quantification were 0.008 mU/L, 0.1 ng/mL and 5.00 ng/mL for thyroid stimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4), respectively.

Serum levels of Total T4 and TSH were considered to be unaffected by treatment with the test material up to 1000 mg/kg bw/day. Furthermore, serum total T3 level at 100 and 300 mg/kg bw/day were unaffected by treatment with test material. At 1000 mg/kg bw/day, decreased serum levels of total T3 were noted. The mean was determined to be 0.372 ng/mL, which was 0.83x of control for which mean was 0.446 ng/mL. As these values remained within the historical control data (HCD) range and there were no concomitant histopathological changes observed in the thyroid, the observed increase in the study was considered non-adverse.

HCD T3: 0.424 ng/mL, range P.2.5-P97.5: 0.270-0.683 ng/mL

Description (incidence and severity):

There were no test material-related alterations in thyroid gland weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be related to treatment with the test material.
There were no test material-related gross observations in the thyroid glands.
Findings that were noted among control and/or treated animals were considered to be unrelated to treatment with the test material, as they remained within the range of biological variation for rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic observations in the thyroid glands.
The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Early or late resorptions:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Dead fetuses:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females except one control and one mid dose (300 mg/kg bw/day) were gravid and had litters with live fetuses.
See table in "Any other information on results incl. tables"

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

corrected for water content

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test material-related effects on fetal body weights (both sexes) noted.
See table in "Any other information on results incl. tables"

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See table in "Any other information on results incl. tables"

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment with the test material.
See table in "Any other information on results incl. tables"

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test material-related effects on litter size of any group.
See table in "Any other information on results incl. tables"

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no test material-related effects on fetal anogenital distance (both sexes) noted.
See table in "Any other information on results incl. tables"

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related external malformations and variations were recorded.
One fetus from the low-dose group (100 mg/kg bw/day) presented with omphalocele
whereby a portion of intestines protruded. Based on its single occurrence this was considered not to be related to treatment with the test material. No other external malformations occurred, and no external variations were recorded.
Incidental external findings not otherwise classified as malformations or variations were not
observed in this study.
See table in "Any other information on results incl. tables"

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related skeletal malformations and variations were recorded.
Two fetuses were observed with skeletal malformations. A fetus from the high-dose group (1000 mg/kg bw/day) had sternoschisis and a control fetus missed half of a lumbar centrum. The singular occurrence of these malformations and/or occurrence in a control fetus did not indicate a relationship with the test material and as such they were ruled as chance findings.
All recorded skeletal variations occurred in a diverse array of skeletal structures across all groups. Also, these variations were either scored infrequently, were comparable to the control group or occurred in the absence of a dose-relationship. Therefore, these skeletal variations were considered not to be related to treatment with the test material.
See table in "Any other information on results incl. tables"

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related visceral malformations and variations were recorded.
Two fetuses were observed with visceral malformations and both were from the low-dose group (100 mg/kg bw/day). The fetus with omphalocele also had an interrupted aortic arch, ventricular septal defect and absent tracheal cartilage rings. The other fetus had transposition of the great vessels and showed a ventricular septal defect. Due to the exclusive occurrence of these findings in single fetuses of the low-dose group, they were considered not to be related to treatment with the test material.
Visceral variations were limited to supernumerary liver lobes, convoluted and dilated ureters, and absent renal papilla. The low incidences and group distribution of these findings did not indicate a relationship with the treatment of the test material.
Incidental visceral findings not otherwise classified as malformations or variations were not observed in this study.
See table in "Any other information on results incl. tables"

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

corrected for water content

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Dose formulation analyses

Accuracy
The concentrations analyzed in the formulations of Group 2 were 52.7, 51.2, 56.3, 55.3, 55.7, and 56.8 mg/mL and for Group 3 were 163, 170, 146, 177, 173, and 183 mg/mL. They were in agreement with the target concentrations of 59 mg/mL for Group 2 and 177 mg/mL for group 3, as the mean sample concentration results were withing or equal to 90-110% of the target concentration.

A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1. It was considered not to represent test material since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.00048%.

Homogeneity
The formulations of Groups 2 and 3 were homogeneous as the coefficient variation was 4.0 and 7.0 for Group 2 and 3, respectively.

 

 

Dose level (mg/kg bw/day)	0	100	300	1000
Pregnant/total dams	21/22	22/22	21/22	22/22
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	0
Dams with live fetuses	21	22	21	22
Corpora lutea (mean number)	12.6	13.5	13.6	12.7
Implantations (mean number)	12.0	12.4	12.9	11.8
Resorptions (mean number) - Early - Late - Total (early + late)	0.6 0.0 0.6	0.7 0.0 0.7	0.8 0.0 0.8	0.5 0.0 0.5
Pre-implantation loss (%)	4.88	7.96	4.84	6.55
Post-implantation loss (%)	4.64	6.03	6.12	3.66
Dead fetuses (mean number)	0.0	0.0	0.0	0.0
Mean body weight on day 21	330.7	334.8	330.8	328.5
Mean adjusted body weight gain (6-abw)	25.99	24.98	21.71	24.55
Gravid uterine weight (g)	77.96	80.70	83.94	79.09
Live offspring (mean number)	11.4	11.6	12.2	11.3
Sex ratio (male%)	50.82	46.36	50.00	50.89
Mean fetal/pup body weight males (g)	5.207	5.289	5.297	5.308
Mean fetal body weight females (g)	4.960	5.044	4.998	5.105
Mean fetal body weight (sexes combined) (g)	5.087	5.152	5.155	5.213
Mean anogenital distance males (mm)	3.11	3.14	3.11	3.05
Mean anogenital distance females (mm)	1.41	1.37	1.40	1.40
Mean normalized anogenital distance males	1.794	1.805	1.782	1.751
Mean normalized anogenital distance females	0.828	0.796	0.822	0.813
Malformations number and % of fetuses number of litters - External     - Visceral     - Skeletal	0 (0.0) 0   0 (0.0) 0   1 (0.95) 1	1 (0.41) 1   2 (1.33) 2   0 (0.0) 0	0 (0.0) 0   0 (0.0) 0   0 (0.0) 0	0 (0.0) 0   0 (0.0) 0   1 (0.91) 1
Variations number and % of fetuses number of litters -External     -Visceral     -Skeletal	0 (0.0) 0   5 (3.97) 3   83 (70.32) 21	0 (0.0) 0   11 (8.46) 8   93 (71.81) 22	0 (0.0) 0   13 (11.37) 8   79 (64.27) 19	0(0.0) 0   7 (6.41) 5   84 (67.53) 21

 

Applicant's summary and conclusion

Conclusions:

A developmental toxicity study was performed in Wistar Han rats according to OECD TG 414 and in accordance with GLP principles. The maternal and developmental NOAEL for the substance were both established as being at least 1000 mg/kg bw/day based on the absence of adverse effects.

Executive summary:

A developmental toxicity study was performed according to OECD TG 414 and in accordance with GLP principles. The objectives of this study were to determine the potential of the substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on the results of the Dose Range Finder (Test Facility Sponsor No. 20297966).

Chemical analyses of formulations were conducted once during the study and confirmed that formulations of test material in Water (Elix) were prepared accurately and homogenously. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH)), organ weights (thyroid gland), macroscopic examination, microscopic examination (thyroid gland) and uterine contents (including corpora lutea, implantation sites, pre- and postimplantation loss and number of live and dead fetuses).
In addition, the following parameters were determined for the F1-generation: fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.
One female at 300 mg/kg bw/day was sacrificed in extremis on Day 9 post-coitum, which was considered not to be related to treatment with the test material.
Incidental abnormal breathing sounds were noted in few females of all dose groups (without a dose relationship) and serum total T3 levels were decreased at 1000 mg/kg bw/day. Clinical signs were considered non-adverse. As the serum total T3 levels remained within the historical control data (HCD) range and there were no concomitant histopathological changes observed in the thyroid, the observed increase in the study was considered non-adverse.
No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., body weight, food consumption, thyroid hormone levels (T4, TSH), thyroid gland weights, macroscopic evaluation, microscopic evaluation of the thyroid gland and uterine contents including corpora lutea implantation sites and pre- and postimplantation loss).

No developmental toxicity was observed up to the highest dose level tested (i.e.,1000 mg/kg bw/day).
In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for the substance were both established as being at least 1000 mg/kg bw/day.