Aluminium tridiethylphosphinate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2012 - 18 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Batch No.: DEH2029007
Purity: 96.9%
Expiry Date: 13.6.2016

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 12-13 weeks old
- Weight at study initiation: males: 311 - 350 g - (mean: 329 g, ± 20% = 263.20 – 394.80 g); females: 200 - 239 g - (mean: 217 g, ± 20% = 173.42 – 260.12 g)
- Fasting period before study: none
- Housing: - Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0856)
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 28 August 2012 To: 29 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylenglycol 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was weighed into a tarred plastic vial on a precision balance.
The test item was suspended in Polyethylenglycol 400.
The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg BW
- Lot/batch no. (if required): 1101401034
- Purity: 100 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses (8 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study (18 samples in total).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high, medium, and low dose formulations (6 samples).
All formulation samples were stored at -20° C and were analysed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH. The procedures followed for the sample analysis were mentioned in the phase plan (BSL phase study no. 124188).

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused: Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. The subsequent morning and the next morning onwards, the vaginal smear of each female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number
- M/F ratio per cage: 1:2
- Length of cohabitation: maximum of 14 days
- Further matings after two unsuccessful attempts: N.A.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: None

Duration of treatment / exposure:

The test item is orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5 to gestation day (GD) 19

Frequency of treatment:

Once per day. 7 days per week

Duration of test:

On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section.

No. of animals per sex per dose:

The 4 groups comprised 25 female Wistar rats, except the HD group where only 24 animals were integrated into this study. This was due to the total amount of pregnant females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except on the day of their arrival.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No


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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination, the dam (presumedly pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.

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:

- 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

Statistics:

A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Chi-square test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: clinical symtoms

Details on maternal toxic effects:
No mortalities were recorded in the C group or any treatment group.
Clinical symptoms were found in all groups (C and treatment groups) and some of them could be found increased in MD and HD group. The most prominent were diarrhea (9/24 HD animals, 4/25 C animals), diarrhea directly after administration (8/24 HD animals, 2/25 C animals), moving the bedding immediately after administration (20/24 HD animals, 8/25 MD animals, 0/25 C animals), slight piloerection (6/24 HD animals, 4/25 MD animals, 0/25 C animals), slight piloerection immediately after administration (8/24 HD animals, 5/25 MD animals, 0/25 C animals), slight salivation immediately after administration (4/24 HD animals, 2/25 MD animals, 1/25 C animals). These symptoms could be concerned with a toxicological relevance in HD group (mostly due to diarrhoea) and with general discomfort of the animals in MD and HD groups. However, since no influence on weight development was observed a clear toxicological relevance cannot be assumed.
Throughout the treatment period, body weights and body weight gain were within the normal range of variation for this strain and no difference could be found between C and treatment groups.
In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in all groups. In treatment groups there was a slightly increased food consumption which was not dose-dependent over all groups.
The statistical analysis of prenatal data revealed no significant effect on each of the parameters. An increased percentage of pre-implantation loss in the LD group is assumed to be incidental, since neither MD nor HD group were affected. An increased percentage of post-implantation loss in the HD group is not assumed to be test item related due to the missing statistical significance combined with the high standard deviation
No treatment related change was observed during the macroscopic observation. Only incidental findings were recognized.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no significant effect up to the highest dose level

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistical significant effect was found in treatment groups of litter data such as mean litter weight, number of males, number of females, total number of pubs, total litter weight, male litter weight, and female litter weight. No influence of the test item on any of the parameters was observed.
Few gross external abnormalities were seen among the control and treatment groups but none of them was statistical significant and all of them are assumed to be incidental.
Skeletal examination of the Alizarin red stained foetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparably lower in treated groups when compared to the control group.
Several abnormalties were increased when compared to C group which were either statistically significant or in a range that it could be of toxicological relevance. These are as follows:
A 14th rudimentary rib (right side; p<0.05 C vs HD)) was found in 13 fetuses of HD group and in 5 fetuses of C group. The incidences were 3.73 % (C) and 12.26 % (HD). The number of litters affected were 4 (C) and 8 (HD). This could be test item related but cannot be assumed as malformation but as variation. Hence, a toxicological relevance is not assumed.
Increased fused frontal and parietal bones were recognized at the skull in treatment groups and the incidences of C vs LD groups were significantly different (p<0.05). In particular 2 incidences were counted in C group as well as 7 (LD), 6 (MD), and 6 (HD) incidences which were counted in treatment group. The percentage incidence of abnormalties was 1.49 % (C), 6.31 % (LD), 5.77 % (MD), and 5.66 % (HD). The number of litters with at least 1 incidence was 2 in C, LD, MD, and HD groups, respectively. Since there was no dose-dependency in percentage of incidences and since the same number of litters was affected, this finding is not assumed to be treatment related but incidental.
 
An incomplete ossification of the interparietal (skull) was significantly increased (p<0.01) in HD group when compared to C group. A percentage of 61.32 % was calculated as abnormal in HD group when compared to 44.03 % in C group. Furthermore, 16 (HD) and 20 (C) litters were affected. Due to the decrease in affected litters, this is not assumed to be test item related.
An incomplete ossification was also mentioned for frontal (skull) in treatment groups when compared to C group (not statistical significant). 14.15 % affected fetuses (HD), 10.58 % (MD), 9.91 % (LD), and 7.46 % (C) were calculated. The number of affected litters was 7 (C), 5 (LD), 8(MD), and 9 (HD). A test item relation is not assumed. No statistical significance could be calculated and the number of affected litters did not follow a dose response and showed only slight and variable changes.
An increased incomplete ossification was mentioned for the parietal (skull) for HD group when compared to C group (p<0.01). 48.11 % (HD) and 30.6 % (C) affected fetuses were counted. The number of affected litters was 15 in both groups. Hence, a test item relation is not assumed but an incidental case.
Several other differences among treatment groups and C groups were statistically significant but are not assumed to be treatment related and/or of toxicological relevance (due to lack of dose dependency and/or due to a higher incidence in C group).
Internal examinations of the fetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups including the control.
A slightly increased percentage of concerned fetuses could be found for hemorrhigoc adrenal glands (both sides). 10.2 % (HD), 7.45 % (MD), 7.77 % (LD), and 4.92% (C) were counted / calculated. The number of affeceted litters was 3 (C), 7 (LD), 5 (MD), and 8 (HD). A statistical significance was not found and a dose-dependency is not present. Hence, it is not assumed that these slight increases of hemorrhagic adrenals are of test item related.
A statistical significant increase could be found for hemorrhagic lungs when compared C with MD group. However, no dose dependency could be found and in HD group just 1 incidence could be counted. Hence, this is not assumed to be treatment related.
Convoluted ureters (both sides) were found clearly and statistically (LD; HD vs C p<0.001; MD vs C p<0.01) incrased in treatment groups when compared to C group. In particular: fetal incidences of 3.31. % (C), 18.45 % (LD), 17.65 % (MD), and 22.83 % (HD) were evaluated. The amount of litters with at least 1 incidence was 4 (C), 12 (LD and MD), and 14 (HD). Although not dose dependent, this is assumed to be test item related. However, since the convoluted ureter is assumed to be a variation and no malformation, this is not assumed to be of toxicological relevance.
An increased percentage of dilated ureters could be found on both sides (not statistical significant). In particular, 2.46 % (C), 6.80 % (LD), 5.32 % (MD), and 8.16 % of all respective fetuses were affected. Furthermore, the number of affected litters was 3 (C) and 5 (LD, MD, HD). This coincides with a slight increase in dilated renal pelvis where 15.22 % (HD) and 8.26 % (C) of all fetuses were affected. However, no statistical significance was calculated and this finding is mostly interpreted as variation. Hence, a toxicological relevance is not assumed.
An increased percentage of a thin ventricular wall was recored in HD group (5.1 % of all fetuses) when compared to C group (1.64 % of all fetuses). The numbers of affected litters were 2 (C), 3 (LD), 1 (MD), and 4 (HD). Since no statistical significance is calculated and since the number of affected litters did not follow a dose response this is not assumed to be test item related but incidental.
Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls. The statisitical analysis of the data mostly revealed no statistical significant and biological relevant differences in the number of incidences in treated groups as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature. The observed significant changes are not considered to be test item related, since only MD and LD groups were concerned.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

The registration substance was investigated for its developmental toxicity in rats according to the OECD Guideline 414. NOAEL of 1000 mg/kg/day was obtained for maternal as well as for fetal toxicity. No indication of developmental toxicity was found in rats.

Executive summary:

The registration substance was investigated for its developmental toxicity in rats according to the OECD Guideline 414. The registration substance was suspended in PEG and given to pregnant rats per gavage at doses of 0, 100, 300 and 1000 mg/kg/day from GD 5 -19. On day GD20 cesarian section was performed. No significant maternal toxicity was found up to the highest dose and no significant fetal toxicity was found up to the highest dose. NOAEL of 1000 mg/kg/day was obtained for maternal as well as for fetal toxicity. No indication of developmental toxicity was found in rats.