Amoxicillin

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD 422. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate.

Cas Number:

61336-70-7

Molecular formula:

C16H25N3O8S

IUPAC Name:

(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate.

Constituent 2

Reference substance name:

amoxicillin trihydrate

IUPAC Name:

amoxicillin trihydrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Amoxicillin trihydrate
- Physical state: White powder
- Stability under test conditions: The test item was found to be stable over a 7 day period (86 %).
- Storage condition of test material: Room temperature, storage in a plastic, glass, or metal container.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

EST ANIMALS
- Source: Husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok.
- Age at study initiation: 12-14 weeks old.
- Weight at study initiation: Males 390.2-410.9 g and females 247.7-248 g
- Fasting period before study: No
- Housing: Same conditions during acclimatisation and experiment, air-conditioned rooms.
- Diet:Altromin 1324 P TPF ad libitum.
- Water: Tap water ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24ºC
- Humidity (%): 35-57 %
- Air changes: 13times/hour
- Photoperiod: 12 hours dark/12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

(0.5 % CMC sodium salt)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test item in vehicle (0.5% Carboxymethylcellulose sodium salt) were prepared once a week (the stability of Amoxicillin in a vehicle in a temperature 4-8ºC during 7-days period was evaluated). During administration, suspensions were kept at room temperature and they were thoroughly mixed.

DIET PREPARATION
- Rate of preparation of diet (frequency): Standard diet was given.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle control was selected on the basis of solubility of the test item in water.
- Concentration in vehicle: 40mg/mL, 140 mg/mL, 490 mg/mL
- Amount of vehicle (if gavage): 0.5 mL/100 gr
- Lot/batch no. (if required): 1296363
- Other: Name: Carboxymethylcellulose sodium salt, ultra high viscosity, highly purified, purchased from Sigma Aldrich.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical concentration was measured using a validated HPLC method with DAD detector. The analytical method was validated (Linearity= 1-500 mg/L; Specifity was evaluated with fortitied samples and the matrix. No signal was overlapping. Recovery=92-102.41 %. Precision= Assessed with repetability, RSD= 0.055-0.27 %. LOQ= 1mg/L. LOD= 0.25 mg/L).

The analytical concentrations were measured weekly and ranged as follows:
35.8 – 46.4 mg/mL for the concentration of 40 mg/mL in vehicle (89.5 – 115.9%)
129.0 – 163.9mg/mL for the concentration of 140 mg/mL in vehicle (92.1 – 117.1 %)
442.3 – 574.4 mg/mL for the concentration of 200 mg/mL in vehicle (90.3 – 117.2%).
The results were within the range of 80 -120%.

The test item was stable in vehicle, minimum for one week: Initial concentration = 40 mg/L, after 7 days 34.6-40.2 mg/L.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 or 1:2
- Length of cohabitation: . The average number of days needed for copulation (the day when males and females were placed together was day 0) was 1.5 in the control group, 1.3 in group 1, 1.0 in group 2 and 1.2 in group 3.
- There was no need of replacement of males for unsuccessful pairing.
- Verification of same strain and source of both sexes: [yes / no (explain)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear]referred to as day 0 of pregnancy

Duration of treatment / exposure:

Males were dosed for 28 days: 14 days pre-mating and 14 days during mating.
Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 60 days in total, 53 average).

Frequency of treatment:

Once daily seven days a week.

Duration of test:

66 days.

No. of animals per sex per dose:

10 males/dose
12 females/dose
10 animals/sexe/dose in the satellite groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for the study were selected on the basis of bibliographic and registrant information.
- Rationale for animal assignment: Animals were randomly assigned to the groups.
- Other: in the study there were satellite groups, however since they were not mated, findings are considered irrelevant for the reproductive/developmental screening half.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily.

BODY WEIGHT: Yes
Time schedule for examinations: Twice a week during the entire experiment.
Body weights of females were measured twice a week before gestation, on days 0, 7, 14, and 20 of gestation, within 24 hours of parturition (day 0 or 1 post-partum), and day 4, 7, 10 and 13 post-partum. Body weights of pups were measured on day 0 (within 24 hours of parturition), 4, 7 and 13 post-partum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 4 and 13
- Organs examined: The animals were subjected to detailed gross examination which involved observations of the external body surface, all natural apertures and the cranial, thoracic, and abdominal cavities with their content. Special attention was paid to the reproductive system and thyroid glands. Absolute and relative weights of the brain, thymus, heart, liver, spleen, kidneys, and adrenals of 5 adult males and 5 adult females from each group were determined. Additionally, absolute and relative weights of the testes, epididymides, accessory sex organs (prostate with the seminal vesicles and coagulating glands), thyroid glands and ovaries of all adult animals were determined with the exception of the female no. 17 in group 3, in which only the absolute and relative weights of the thyroid glands was determined, without the weights of the ovaries, as it was non-pregnant.
The histopathological examination of the following organs and tissues collected from five adult males and females from group 0, 0 SAT, 3 and 3 SAT was conducted: brain with the cerebellum and pons, pituitary gland, eyeball with optic nerve, lymph nodes, spinal cord, muscle with the peripheral nerve, trachea, thyroid with the parathyroids, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, spleen, lungs, heart, thymus, kidneys, adrenals, urinary bladder, ovaries, uterus with the cervix and horns, vagina, mammary glands, testes, epididymides, accessory sex organs (prostate with the seminal vesicles and coagulating glands), and all organs with gross lesions. The following organs and tissues of animals were examined: testes, epididymides and accessory sex organs (prostate, vesicular and coagulating glands) from all adult males; ovaries from all adult females; thyroid glands from all adult rats.

Please refer to Endpoint specific 7.5.1 For repeated dose toxicity parameters.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes (separately in the left and the right ovary)
- Number of implantations: Yes, sites were also determined separately in the left and the right horn of the uterus
- Number of early resorptions: No
- Number of late resorptions: No
- Other: ovary weights, histological examination of ovaries, uterus with the cervix and horns, vagina.

Fetal examinations:

- External examinations: Yes: all per litter, they were carefully examined for gross abnormalities. The examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content
- Soft tissue examinations: Yes: all per litter The tissues and internal organs were examined.
- Skeletal examinations: The gross examination included observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content .
- Head examinations: Yes: all per litter

Other: The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined once from
- two pups per litter on day 4 after birth,
- two pups per litter on day 13 after birth.
- The anogenital distance was measured on day 4 afterbirth.
- The number of nipple/areolae were determined on day 13 after birth.

Statistics:

The treated groups, i.e. groups 1, 2, and 3 were compared to the control group.
The clinical results were statistically analyzed using the one-way analysis of variance and the Dunnet’s test and Student’s t-test (group 0 SAT, 3 SAT) (p ≤ 0.05).
The clinical-chemical results were statistically analyzed using the one-way analysis of variance and Dunnet’s test (p ≤ 0.05).
The results, i.e. absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test (p≤0.05).

Indices:

The following elements were determined for each group:
Fertility of parental animals:
Mating index for males:
the number of males showing evidence of copulation/the total number of mated males x 100
Mating index for females:
the number of sperm-positive females/the total number of mated females x 100
Fertility index for males:
the number of males that impregnated females/the total number of mated males x 100
Fertility index for females:
the number of pregnant females/the total number of sperm-positive females x 100
Pregnancy index:
the number of litters with live births/the number of pregnant females x 100.

Offspring survival:
Index of live births:
the total number of live newborns/the total number of newborns x 100
Index of 4-day survival:
the number of live sucklings after 4 days/the number of live newborns x 100
Index for 13-day survival:
the number of live sucklings after 13 days / (the number of live newborns – number of sucklings transferred to examination in day 4) x 100 .
The following parameters were also assessed:
the average number of days required for copulation (the day when males and females were placed together was day 0);
the length of gestation.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No adverse dose-related effects were found (for more details please refer to endpoint specific 7.5.1 repeated dose toxicity).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

2 450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

2 450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were not observed any pathological changes and malformations. The macroscopic evaluation of the offspring did not reveal any lesions or malformations in live-born and stillborn pups.
There were no statistically significant changes in average body weights of the pups per litter between the treated and the control groups.
There were no statistically significant changes in AGD measurment in male and female pups between the treated and the control groups.
There were no statistically significant changes in number of nipples / areolae in male pups on 13th day of life between the treated and the control groups.
The results of the hormonal examinations (TT4) in pups 13 days after birth did not show statistically significant changes.
The following clinical observations were recorded
temporary hematoma on the neck in one male from group 2;
temporary hematoma on the back in one male from group 0 and one male from group 2;
temporary hematoma on the head in one male from group 0 and one male from group 2;
temporary hematoma on the hindlimb in two male from group 2, and one male from group 3;
temporary scab on the left ear in one male from group 0;
thinning hair in three males and two females from group 0, and four males and two females from group 1;
smaller body in one male and one female from group 0;
smaller and livid body in one male from group 0.
However considering the distribution in the control and test item-treated groups, considering the severity and persitence of such findings, they are considered not to be related to the test item.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1. Amoxicillin trihydrate. Mating of parental animals – list of results

  

Parameter	GROUP
	0	1	2	3
Number of males to mating	10	10	10	10
Number of females to mating	12	12	12	12
Number of males which copulated	10	10	10	10
Number of females which were mated	12	12	12	12
Average number of days to copulation	1.5	1.3	1.0	1.2
Number of fertilized females	12	12	12	11
Number of pregnant females	12	12	12	11
Number of females which delivered	12	12	12	11
Number of not pregnant females	0	0	0	1
Number of females which delivered offspring	12	12	12	11
Number of females which delivered live offspring	12	12	12	11

 

Table 2. Amoxicillin trihydrate. Indices concerning fertility of parental animals

 

Parameter	GROUP
	0	1	2	3
Mating index for males	100.0	100.0	100.0	100.0
Mating index for females	100.0	100.0	100.0	100.0
Fertility index for males	100.0	100.0	100.0	90.0
Fertility index for females	100.0	100.0	100.0	91.7
Pregnancy index	100.0	100.0	100.0	100.0
Length of gestation	22.3 ± 0.5	22.4 ± 0.5	22.2 ± 0.4	22.6 ± 0.5

 

Table 3. Amoxicillin trihydrate. Average number of pups in litter

 

Parameter	GROUP
	0	1	2	3
Total number of pups in litter	11.8 ± 2.3	12.3 ± 1.6	12.2 ± 2.2	12.3 ± 2.1
Number of live births in litter	11.7 ± 2.2	12.3 ± 1.5	11.8 ± 3.0	11.7 ± 2.4
Number of pups in litter, min.– max.	9 - 15	8 - 14	9 - 15	7 - 15
Percentage of males in litter	48.6	51.4	52.7	50.4
Percentage of females in litter	51.4	48.6	47.3	49.6

Table 4. Amoxicillin trihydrate. Indices connected with survival of offspring

Parameter	GROUP
	0	1 2	2	3
Index of live births	98.6	99.3	96.6	95.6
Index of 4-day survival	97.1	98.0	97.2	99.2
Index of 13-day survival	95.8	97.6	96.6	99.1
Mortality [%]	4.2	2.4	3.4	0.9

 

Table 5. Amoxicillin trihydrate. Average AGD [mm] in male pups in litter on 4th day of life (statistically analyzed)

 

Day	GROUP
	0 n=12			1 n=12			2 n=12			3 n=11
4	5.16	±	0.43	5.13	±	0.10	5.20	±	0.16	5.13	±	0.16

 

 

Table 6. Amoxicillin trihydrate. Average AGD [mm] in female pups in litter on 4th day of life(statistically analyzed)

 

Day	GROUP
	0 n=12			1 n=12			2 n=12			3 n=11
4	3.31	±	0.16	3.28	±	0.11	3.28	±	0.15	3.25	±	0.15

 

Table 7. Amoxicillin trihydrate. Average number of nipples / areolae in male pups in litter on 13th day of life(statistically analyzed)

 

Day	GROUP
	0 n=12			1 n=12			2 n=12			3 n=11
13	0.10	±	0.25	0.02	±	0.06	0.09	±	0.15	0.01	±	0.04

There were 571 pups. 10 animals were eaten by mother. Among the remaining 561 animals, 14 of them were stillborn pups (2 females in the control group, 1 male in group 1, 4 males and 1 female in group 2, 3 males and 3 females in group 3), whereas 3 died during observation after the birth (1 female in group 0, and 2 males in group 1).The macroscopic examination of the all pups (without pups eaten by mothers) did not reveal any pathological changesor malformations.

Applicant's summary and conclusion

Conclusions:

The NOAEL of the parental toxicity and developmental toxicity for amoxicillin trihydrate in rats by oral route was determined to be equal or higher than 2450 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted with amoxicillin trihydrate according to OECD 422 under GLP conditions. Twuelve to fourteen weeks old Wistar rats were exposed to amoxicillin trihydrate at concentrations of 0 (vehicle control), 200, 700 and 2450 mg/kg-bw/day by oral gavage in 0.5 % carboxymethyl cellulose sodium salt. There were 10 males and 12 females in each group. The test item was administered for 28 days(14 days premating, during mating and post mating period) to males and for 51-60 days to females (premating, conception, pregnancy and at least 13 days after delivery, average 53 days). All offspring was weighed on day 4 after birth, that day blood from 2 pups was collected for analysis of total TT4 thyroid hormone, the anogenital distance and the number of nipple/areolae was measured on day 13.The euthanized and the dead animals were subjected to the post-mortem examinations. Their tissues and internal organs were examined. The treatment of male and female rats with amoxicillin trihydrate at dose levels up to 2450 mg/kg-bw/day by repeated oral (gavage) administration revealed no parental toxicity nor adverse effects on the reproductive performance or fertility. There were no indications of developmental effects in the offspring. The no-observed-adverse-effect-level (NOAEL) of amoxicillin trihydrate was determined to be 2450 mg/kg bw/day for parental, reproductive and developmental toxicity.