Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Toxicity to reproduction

Administrative data

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar 1984 - May 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: US EPA 163.83-4

Version / remarks:

1978

Principles of method if other than guideline:

To assess the effect of the test substance upon the growth and reproduction of multiple generations in the rat, it was mixed with granulated feed, pelleted and administered orally to the F0 generation of Wistar / HAN rats during an 80-day prepairing and also during the pairing, gestation and lactation periods for breeding of the F1A and F1B litters. Following the weaning of the F1B litters on day 21 post partum, the pups were reared for a further seven days on the test diet. Selection of the F1 parents and actual treatment of the F1 generation was considered to have commenced when they were approximately four weeks of age. The test article was administered during a 101-day prepairing period and also during the pairing, gestation and lactation periods for breeding of the F2A and F2B litters. The control group received the same diet without the test article.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

EC Number:

278-636-5

EC Name:

Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Cas Number:

77182-82-2

Molecular formula:

C5H12NO4P.H3N

IUPAC Name:

ammonium 2-amino-4-[hydroxy(methyl)phosphoryl]butanoate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HAN, outbred, SPF quality

Details on species / strain selection:

This system has been selected as standard rodent species (according to guidelines) and is internationally recognized for this type of investigation.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM, Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: 130 - 177 g (males), 112 - 149 g (females)
- Housing: individually
- Diet (e.g. ad libitum): pelleted standard Kliba 343, rat / mouse maintenance diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at least every 2 weeks
- Mixing appropriate amounts with (Type of food): standard Kliba 343, rat / mouse maintenance diet
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 9 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test article, its content, homogeneity and stability in the food pellets were performed before starting the test, content and homogeneity at the start of pairing and at the end of gestation / start of lactation periods for breeding of F1A, F2A and F2B generations. The analyses were performed in the Analytical Laboratories of RCC according to an analytical method supplied by the sponsor.

Duration of treatment / exposure:

- F0 parent animals: The test article was administered during an 80-day prepairing- and also during pairing-, gestation- and lactation periods for breeding the F1A and F1B litters / pups.
- F1 parent animals (originated from F1B litters / pups): Following weaning on day 21 and the removal of the dams, the F1B pups were reared for a further seven days on the test diet. At this date, 26 male and 26 female pups were randomly selected to form the F1 parent animals. Actual treatment (food consumption) of the F1 males and females was considered to have commenced when they were approximately four weeks of age. The test article was administered during a 101-day prepairing period and also during pairing, gestation- and lactation periods for breeding of the F2A and F2B litters / pups.

Frequency of treatment:

daily

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 18 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

40 ppm

Doses / concentrations 3

Dose / conc.:

120 ppm

Doses / concentrations 4

Dose / conc.:

360 ppm

No. of animals per sex per dose:

F0 generation: 30
F1 generation: 26

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dosages were based upon the results of the preliminary study, in which 500 ppm in the diet caused a high postimplantation loss of 43.3 %, which was shown to be related only to effects in the females, was considered to be excessively high for use in a multiple generation study.

Examinations

Parental animals: Observations and examinations:

MORTALITY / VIABILITY: Yes
- Time schedule: at least twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: at keast twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: weekly
- The food conversion ratios and consumption of the test article (in mg/kg bw/day) were calculated.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
testis weight, prostate weight and seminal vesicles weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 & F2 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals shortly after the F1B pups were weaned
- Maternal animals: All surviving animals shortly after the F1B pups were weaned

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination: all gross lesions, aorta, adrenals, bone (with marrow, vertebra), brain (cerebrum, cerebellum, brainstem), eyes and the contiguous Harderian glands, heart, intestinal tract (esophagus, stomach, small intestine (duodenum, jejunum), large intestine (cecum, colon)), kidneys, liver (with at least two lobes), lung (with mainstream bronchi), lymph nodes (cervical, mesenteric), mammary gland, ovaries, pancreas, pituitary, porstate and accessory glands, salivary gland (submaxillary), seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord (two levels), spleen, stomach, testes with epididymides, thymus in pups or where present, thyroids (with parathyroids if possible), tongue, trachea, urinary bladder, uterus (corpus and cervix), nasal cavity / turbinates, macroscopical changes

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.

GROSS NECROPSY
- Gross necropsy consisted of: brain (including entire brain stem), adrenals, spleen, uterus, pituitary, thymus, testes or ovaries, heart, kidneys, liver

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues were prepared for microscopic examination: all gross lesions, aorta, adrenals, bone (with marrow, vertebra), brain (cerebrum, cerebellum, brainstem), eyes and the contiguous Harderian glands, heart, intestinal tract (esophagus, stomach, small intestine (duodenum, jejunum), large intestine (cecum, colon)), kidneys, liver (with at least two lobes), lung (with mainstream bronchi), lymph nodes (cervical, mesenteric), mammary gland, ovaries, pancreas, pituitary, porstate and accessory glands, salivary gland (submaxillary), seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord (two levels), spleen, stomach, testes with epididymides, thymus in pups or where present, thyroids (with parathyroids if possible), tongue, trachea, urinary bladder, uterus (corpus and cervix), nasal cavity / turbinates, macroscopical changes

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and reproduction data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- 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 between the treated groups and the control groups.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2 x 2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related signs of toxicity or clinical symptoms were evident in any animal of any dose group in any generation.
The isolated deviations and variations in behavioral and appearance noted in a few animals were considered to be spontaneous in origin and common in rats of this strain and age.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Throughout this multiple generation reproduction study on the test substance, no test article-related deaths occurred in the parent animals of either generation.
One group 3 (120 ppm) male lost body weight rapidly and died on day 8 after the pairing period for breeding of F1B litters. Additionally, female of group 2 (40 ppm) died during parturition of its F1B litter. These exceptions were considered to be incidental and within the normal mortality-range for animals of this strain and age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males
Before pairing for bredding of F1A litters, the mean body weights of all groups at the beginning of treatment were similar. At the end of the prepairing period of 80 days, no significant differences between the control group and any dose group were evident. After pairing for breeding of F1A litters until pairing for breeding of F1B litters, no test article- or dose-related differences were noted. The body weight gain in all groups was similar. After pairing for breeding of F1B litters until necropsy, the mean body weight gain and the mean body weights on the day of necropsy were similar in all groups. No test article-related or statistically significant differences were evident.

Females
Before pairing for breeding of F1A litters, no test article- or dose-related differences between the dose groups and the control group were noted. The calculation of the body weight gain for hte 80-day prepairing period resulted in similar values of all groups. During the gestation period for breeding of F1A litters, similar mean body weight gain of dams was noted in all groups. During the lactation period for breeding of F1A litters, no test article- or dose-related differences between any dose group and the control group were found. During the gestation period for breeding of F1B litters, no statistically significant differences in the mean body weight gain of dams in all groups were evident. During the lactation period for breeding of F1B litters, no test article-related differences between the dose groups and the control group were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
Before pairing for breeding of F1A litters, the mean food consumption of all test article-treated groups was similar when compared to that of the control group. No treatment-related differences were noted during this prepairing period of 80 days. After pairing for breeding F1A litters until pairing for breeding F1B litters, no treatment-related difference in the mean food consumption of any group was evident. After pairing for breeding F1B litters until necropsy, the mean food consumption of all groups was similar.
Food Conversion of males: The food conversion ratios corresponded to the food consumption and body weights. The food conversion of both generations was similar during all periods.
The nominal dosage levels (test article consumption) of both generations were similar during the entire study.

Females
Before pairing for breeding of F1A litters, no test article-related or statistically significant differences between the control group and any dose group were evident during the prepairing period of 80 days. During the gestation period for breeding of F1A litters, no test article-related or statistically significant differences in the mean food consumption of dams of all groups were evident. During the lactation period for breeding of F1A litters, the comparison of the mean food consumption of dams until day 14 of the lactation period yielded no test article-related or statistically significant differences between the low- and mid-dose group and the control group. The statistically significant reduced food consumption of dams noted in group 4 was due to reduced litter size (number of pups per dam) in this group. During the gestation period for breeding of F1B litters, the mean food consumption of dams of all dose groups was comparable with the values of the controls. During the lactation period for breeding of F1B litters, no test article-related or statistically significant changes in food consumption of the dams of the low- and mid-dose group were evident. The statistically significant reduced food consumption noted of dams of group 4 was considered to be related to the reduced litter size (number of pups per dam) in this group.
Food conversion: The food conversion ratios corresponded to the food consumption and body weights. The food conversion of both generations during all periods was similar. The reduced values noted in the group 4 dams of both generations during the lactation periods was caused by the reduced litter size (number of pups per dam) of these animals.
The nominal dosage levels (test article consumption) of both generations were similar during the entire study. The amount of test article consumed during the different perios varied according to the corresponding food conversion.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

All parameters recorded yielded no evidence of adverse effects on mating performance and fertility except the number of the progeny of group 4 (360 ppm). The mean precoital time, percentage of mating, fertility index, conception rate and gestation index were similar.

Details on results (P0)

No test article-related or statistically significant difference between any group was evident for the following observations: The number of females paired, mated, pregnant, bearing and rearing the pups and also the breeding loss. The number of living pups per dam in group 4 (360 ppm) was significantly reduced when compared with the other dose groups and the controls. This finding was considered to be test article-related.
The mean duration of gestation of groups 1, 2, 3, and 4 was similar.
The behavior of the dams of groups 1, 2, 3, and 4 during parturition and nursing was similar.
One dead pup was found at the first litter check after parturition in groups 2 and 3 and in the control group, respectively. In group 4, four dead pups were found. This findings were considered to be incidental and not treatment-related.
The total and mean number of pups in group 4 were significantly reduced when compared with the other dose groups and the controls. This finding was considered to be test article-related.
No test article-related differences were noted in any dose groups when compared to that of the control group concerning breeding loss.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

120 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced litter size at 360 ppm

Remarks on result:

other: Reproduction

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test article-related signs of toxicity or clinical symptoms were evident in any animal of any dose group in any generation.
The isolated deviations and variations in behavioral and appearance noted in a few animals were considered to be spontaneous in origin and common in rats of this strain and age.

Mortality:

no mortality observed

Description (incidence):

Throughout this multiple generation reproduction study on the test substance, no test article-related deaths occurred in the parent animals of either generation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males
Before pairing for breeding F2A litters, the mean body weights of all groups at initiation of the prepairing period were similar. At the end of the 101-day prepairing period, no significant differences between any group were evident. After pairing for breeding F2A litters until pairing for breeding F2B litters, comparable mean body weights were noted in all groups at start and end of this period. After pairing for breeding F2B litters until necropsy, the mean body weight gain amounted 12 to 17 grams during this period. No statistically significant difference was noted between any group.

Females
Before pairing for breeding of F2A litters, no test article-related or statistically significant differences between the dose groups and the control group were evident during the 101-day prepairing period. During the gestation, lactation period for breeding of F2A and F2B litters, no test article- or dose-related differences in the dose groups were noted when compared to that of the control group. The mean body weight gain in the dams of all groups were similar.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males
Before pairing for breeding F2A litters, the mean food consumption of all treated groups was similar and comparable with the controls. No test article-related changes were noted during the 101-day prepairing period. After pairing for breeding F2A litters, no treatment-related differences in the mean food consumption of any group was evident. After pairing for breeding F2B litters until necropsy, no significant differences were noted between the mean food consumption of the dose groups and the control group.
Food Conversion of males: The food conversion ratios corresponded to the food consumption and body weights. The food conversion of both generations was similar during all periods.
The nominal dosage levels (test article consumption) of both generations were similar during the entire study.

Females
Before pairing for breeding of F2A litters, no test article- or dose-related differences in the mean food consumption of groups 2, 3 or 4 were noted when compared with that of the controls. During the gestation period for breeding of F2A litters, the mean food consumption of dams during the gestation period was similar in all groups. During the lactation period for breeding of F2A litters, no test article-related changes in mean food consumption between the dose groups was noted. During the lactation period for breeding of F2A litters, the significantly reduced mean food consumption values noted in the dams of group 4 was related to the reduced litter size (number of pups per dam) in this group. During the gestation period for breeding of F2B litters, the mean food consumption of the dams of all groups during the gestation period was similar. During the lactation period for breeding of F2B litters, no test article-related change was noted. The statistically significant reduced mean food consumption of the group 4 animals was related to reduced litter size (number of pups per dam) observed in this group.
Food conversion: The food conversion ratios corresponded to the food consumption and body weights. The food conversion of both generations during all periods was similar. The reduced values noted in the group 4 dams of both generations during the lactation periods was caused by the reduced litter size (number of pups per dam) of these animals.
The nominal dosage levels (test article consumption) of both generations were similar during the entire study. The amount of test article consumed during the different perios varied according to the corresponding food conversion.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant weight changes were noted for the kidneys. These were manifested by increased absolute kidney weights in group 3 (120 ppm) and 4 (360 ppm) males in F0 and F1 parents and group 4 (360 ppm) females (F1), increased kidney to body weight ratios which were recorded in groups 3 and 4 males (F1) and females (F0).
Increased organ to brain weight ratios of groups 3 and 4 males (F0 and F1) and group 4 females (F0 and F1) were also noted. No evidence of macroscopical correlation to these findings and no dose-relationship existed.
Other statistically significant absolute and relative organ weight changes were noted, but were considered to be random occurrences.
In the males of the F0 and F1 generations which failed to induce pregnancy in the second mating, no treatment-related changes were noted for seminal vesicles and prostate weights.

Reproductive function / performance (P1)

Reproductive performance:

no effects observed

Description (incidence and severity):

All parameters recorded yielded no evidence of adverse effects on mating performace and fertility except the number of the progeny of group 4 (360 ppm). The percentage mating, fertility index, conception rate and gestation index were similar in all groups.

Details on results (P1)

With the exception of reduced litter size noted in group 4 (360 ppm), no test article-related or statistically significant differences between the control group and any dose group were evident. The number of females paired, mated, pregnant, bearing and rearing the pups and also the breeding loss were similar in all groups.
The mean duration of gestation of the dams of groups 1, 2, 3, and 4 was similar.
The behavior of the dams of groups 1, 2, 3, and 4 during parturition and nursing was similar.
No dead pups were found at the first litter check after parturition in any group.
No differences in the total and mean number of pups in the dose groups 2 and 3 were evident when compared with the control group. In group 4 (360 ppm), the total number of pups and the mean number of pups per dam was reduced. This finding, although not statistically insignificant, was considered to be related to the treatment with the test article.
No test article-related losses were noted in any dose group.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Effect level:

120 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced litter size

Remarks on result:

other: Reproduction

2

Dose descriptor:

NOAEL

Effect level:

360 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Remarks on result:

other: systemic toxicity

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Similar body weights and body weight gain were noted in the pups of all dose groups of all generations. There were no significant or treatment-related changes observed.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant changes in absolute or relative organ weights were noted in F1A and F2A male and female pups.
In F1B and F2B male and female pups, several organs of the group 4 (360 ppm) pups showed slightly but statistically significant increased absolute weights and in some cases, resulting increased relative organ weights. These changes were considered to be related to the increased body weights of the pups of this group.

Details on results (F1)

Similar sex ratios in all groups were noted. No statistically significant differences between the sex ratios of groups 2, 3 and 4 were evident when compared with the respective control group.
The physical development, health conditions, viability and appearance of the pups of all groups were similar; no test article-related differences were noted.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

360 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Remarks on result:

other: systemic toxicity

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Similar body weights and body weight gain were noted in the pups of all dose groups of all generations. There were no significant or treatment-related changes observed.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant changes in absolute or relative organ weights were noted in F1A and F2A male and female pups.
In F1B and F2B male and female pups, several organs of the group 4 (360 ppm) pups showed slightly but statistically significant increased absolute weights and in some cases, resulting increased relative organ weights. These changes were considered to be related to the increased body weights of the pups of this group.

Details on results (F2)

Similar sex ratios in all groups were noted. No statistically significant differences between the sex ratios of groups 2, 3 and 4 were evident when compared with the respective control group.
The physical development, health conditions, viability and appearance of the pups of all groups were similar; no test article-related differences were noted.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

360 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Remarks on result:

other: systemic

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

360 ppm

Treatment related:

not specified

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified