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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
The developmental toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2016 to 30 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 12 - NohSan 8147
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine
EC Number:
941-634-6
Cas Number:
1228284-78-3
Molecular formula:
C10H12Cl3NO
IUPAC Name:
methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine
Test material form:
other: liquid
Details on test material:
- Physical state: Brown liquid
- Expiration date of the lot/batch: Recertification date end May 2017
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 182 to 244 g
- Housing: individually in grid-floor cages over paper lined trays
- Diet and Water (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) were freely available
- Acclimation period: At least two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 23 °C
- Humidity (%): 40 % to 70 %
- Air changes (per hr): not specified (air-conditioned)
- Photoperiod (hrs dark / hrs light): illuminated by fluorescent light set to give a cycle of 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: as a suspension in 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A weighed quantity of test item was added to the final preparation container and approximately 90 % of the final weight of vehicle was added gradually, whilst stirring. The formulation was then stirred and heated to a maximum of 50 °C until a homogeneous mixture was formed. The formulation was allowed to cool before adding vehicle up to the final weight and stirring until mixed. All formulations prepared for Group 4 (formulation concentrations of 12 mg/mL) were homogenised, with the exception of that given on Day 1. The formulation was then divided between amber glass bottles for dosing and stored refrigerated.

Formulations were removed from refrigerated storage and stirred for at least 15 minutes before the start of dosing and until completion of dosing.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples taken from each test item formulation prepared for the first day of dosing were analysed for the test substance using a validated method (BFI030LC (4)) to confirm homogeneity and also achieved concentrations. Having confirmed homogeneity for the first day of dosing for Groups 2 and 3, samples were taken from all test item formulations prepared for use towards the last day of dosing and analysed to confirm concentration only. For Group 4 only, formulations prepared on each occasion were assessed for homogeneity and achieved concentrations. For formulations prepared for Control animals, samples were taken from formulations prepared for the first day of dosing and towards the end of the dosing period and were analysed to confirm absence of test item.

All remaining samples were retained and discarded once the final formulation analysis results were accepted.
Details on mating procedure:
For mating, each female was paired with a male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
Duration of treatment / exposure:
From Day 6 to Day 19 of gestation, inclusive,
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of results from a previous study performed at Sequani (28 Day Oral gavage Stuy Sequani Study Numbers: BFI0239 (1) and Reproduction/Developmental Toxicity Screening Study BFI0319 (2)).

- Species selection rationale: The Han Wistar derived rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain. Background data on the rate of spontaneous malformations have been accumulated by Sequani.

The route of administration was oral (gavage) in accordance with OECD Test Guideline 414, as the oral route is considered a possible route of human exposure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 5 to 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of food consumed by each animal was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative.
The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in neutral buffered formaldehyde.

OTHER:
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
Fetal examinations:
On Day 20 of gestation, live foetuses were killed by rapid cooling, weighed, sexed and examined for external abnormalities.

Approximately 50 % of the live foetuses were allocated to the fixed head examination. All foetuses were briefly placed in 70 % alcohol and subjected to micro-dissection, where the viscera were examined and the foetuses eviscerated. The foetuses allocated to the fixed head examination were decapitated and the heads were fixed in Bouin's fluid for serial sectioning. A coronal section was made through the head of the intact foetuses along the frontal parietal suture and the brain examined. All carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.

Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Other defects were classified as minor abnormalities or variants.

Foetuses with major external or visceral abnormalities were photographed.

For archiving, all foetal heads fixed in Bouin's fluid were stored in neutral buffered formaldehyde and all skeletal specimens were stored in aqueous glycerol with thymol crystals (to prevent fungal growth).
Statistics:
For Quantitative Data: Body weight, cumulative body weight gain from the start of dosing, food intake, terminal body weight, numbers of corpora lutea, implants, live foetuses, dead foetuses, early deaths, late deaths, gravid uterus weight, total litter weight, placental weight and mean foetal weight (sexes separately and combined) were analysed using a parametric ANOVA.
For Percentages: Pre-implantation loss, post-implantation loss, sex ratios and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation.
Maternal Performance: (e.g. the proportion of females with live foetuses at termination, abortions, total resorptions) were analysed by a two-tailed Fisher’s Exact Text, comparing each treated group to the control group.
Foetal Morphology Data: The incidence of foetal malformations and developmental variations (external, visceral and skeletal) were summarised as the proportion of foetuses affected, the proportion of litters affected and the proportion of foetuses affected within each litter. The proportions of litters affected were analysed by the exact version of the CochranArmitage Test. The percentages of foetuses affected within each litter will be analysed by the exact version of the Jonckheere Trend Test. In both cases the tests will be performed in a step-wise manner, where, when a test is significant at the 5% level, the test is repeated after removing the then top dose, until only the control group is left. Tests will be one-sided looking for increase in treated groups versus the control group.
Dunnett’s Test: Dunnett’s Test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s Test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags were presented in the tables of results in the final report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical observations during the study.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg/day, there was a group mean body weight loss from Day 6 to 8 of gestation, resulting in a statistically significantly lower (p<0.05) mean body weight on Day 9 of gestation (6 %) only. However, when adjusted for the weight of the gravid uterus, the mean body weight gain over the dosing period was 29 % lower than the Controls, which was statistically significant (p<0.01).

For animals given 25 mg/kg/day, mean body weight gain was initially lower than the Controls over Days 6 to 7 of gestation. However, from Day 7 of gestation mean body weight gain was similar to the Controls, with overall body weight gain and absolute body weight on Day 20 of gestation, when adjusted for the weight of the gravid uterus, also similar to that of the Control group. The slight effect on body weight gain only at the start of the dosing period is not considered adverse.

At 10 mg/kg/day, body weight and body weight gains were similar to the Controls
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food intake for animals given 60 mg/kg/day was statistically significantly lower than the Controls throughout most of the dosing period, resulting in 14 % lower overall mean food intake for this group.

For animals given 10 or 25 mg/kg/day, food intake was similar to Controls throughout the dosing period.
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
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal necropsy findings that were considered to be related to the substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
With the exception of one female given 25 mg/kg/day, all females were pregnant, resulting in 22, 22, 21 and 22 females with live foetuses in the groups given 0, 10, 25 and 60 mg/kg/day, respectively, on Day 20 of gestation.
For females given 10, 25 and 60 mg/kg/day, the mean number of implantations and the mean number of live foetuses were unaffected by the test item administration.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For females given 10, 25 and 60 mg/kg/day, the incidences of pre- and post-implantation loss were unaffected by the test item administration.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
the mean number of live foetuses were unaffected by the test item administration.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect of test item administration on mean foetal or placental weight, values were similar to the Control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There was no effect of the substance on the incidence of major malformation.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major foetal abnormalities noted in the Controls or the groups given 25 or 60 mg/kg/day. Major foetal abnormalities were noted in two foetuses from two litters in the group given 10 mg/kg/day. These were as follows:
10 mg/kg/day
Female 35 Foetus L7 One or more bifid sternebra

Female 42 Foetus R6 Absent hemicentrum from the thoracic vertebra Absent neural arch from the thoracic vertebra One or more ribs arising from the same neural arch

There was no effect of the test item on the incidence of minor foetal malformations and no adverse effect on the incidence of foetal variations. Although slight differences in foetal variations on the rate of cartilaginous and skeletal development were observed between the groups, achieving slight statistical differences on some occasions, all elements within the cartilage and/or skeleton were considered to be developing normally, following the expected chronological pattern for normal development. Consequently, the changes in incidence of the foetal variations recorded were considered to be transient and not to be indicative of abnormal cartilage or skeletal development.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: rib
skeletal: vertebra
Description (incidence and severity):
Major foetal abnormalities were noted in two foetuses from two litters in the group given 10 mg/kg/day CA5204A. These were as follows:

10 mg/kg/day

Female 35 Foetus L7 One or more bifid sternebra

Female 42 Foetus R6 Absent hemicentrum from the thoracic vertebra
Absent neural arch from the thoracic vertebra
One or more ribs arising from the same neural arch

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 25 mg/kg/day, while the NOAEL for embryo-foetal development was considered to be 60 mg/kg/day.
Executive summary:

Four groups of 22 sexually mature timed-mated female Crl:WI(Han) rats were dosed, once daily by oral gavage, with the test item or vehicle, 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80.  Animals were given 0, 10, 25 or 60 mg/kg/day from Day 6 to Day 19 of gestation, inclusive.

Body weights, food intake and clinical observations were recorded.  All animals were killed on Day 20 of gestation, a necropsy performed and the internal organs examined for gross abnormalities.  The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded.  The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal abnormalities.

There were no deaths and no test item-related clinical observations.  

At 60 mg/kg/day, there was an initial loss in group mean body weight from the start of the dosing period which resulted in an overall body weight gain for the dosing period which was lower than the Controls.  Despite an initial reduction in body weight gain after the start of dosing at 25 mg/kg/day, overall body weight gain in the groups given 10 or 25 mg/kg/day was similar to the Control group.  

At 60 mg/kg/day, mean food intake was reduced throughout most of the dosing period, resulting in overall mean food intake being 14 % lower than Controls.  Food intake at 10 or 25 mg/kg/day was similar to the Controls.

There were no test item-related maternal necropsy findings and there was no effect of CA5204A on uterine or implantation data.  Mean foetal and placental weights and foetal sex ratio were unaffected by CA5204A.  

On this basis the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 25 mg/kg/day, while the NOAEL for embryo-foetal development was considered to be 60 mg/kg/day.

There was no adverse effect of CA5204A on the incidences of major, minor or variant foetal abnormalities.