Aminoguanidinium hydrogen carbonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2014 to 16 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: 10-12 weeks
- Weight at study initiation: The average weight of the males was between 311.0 g and 361.5 g, and the average weight of the females was between 165.5 g and 220 g on Day 1 of the study.
- Housing: Rats were held individually in Polycarbonate type M III cages (floor area of about 800 cm2), with the following exceptions: During overnight matings, male and female mating partners were housed together in Polycarbonate type M III cages and pregnant animals and their litters were housed together until PND 4.
- Diet (e.g. ad libitum): Free access to ground Kliba maintenance diet mouse/rat 'GLP' meal
- Water (e.g. ad libitum): Water Free access to tap-water.
- Acclimation period: At least 6 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 30 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

IN-LIFE DATES: From: 15 April 2014 To: 16 June 2014

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxymethylcellulose suspension in drinking water

Details on exposure:

VEHICLE
-Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
-Dose Volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Method of Formulation – The test substance suspension was weighed in a calibrated beaker depending on the dose group, topped with 1% carboymethylcellulose suspension in drinking water and intensely mixed with a homogenizer. During administration, the preparations were kept homogenous with a magnetic stirrer.

Details on mating procedure:

Following a minimum of 14 days of exposure for males and females, one female was cohabitated with one male of the same treatment group. Detection of mating was confirmed by evidence of sperm in the vaginal smear. This day was designated DAY 0 post-coitum. Male and female rats were separated and housed individually after mating was confirmed. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The average concentration of AGBC in 1% carboxymethylcellulose suspension in drinking water for the three dosing groups was measured using potentiometric tiration with trifluoromethanesulfonic acid. The middle and high dose groups had a concentration of 96% of the expected dose. In the low dose group, the measured concentration was 88% of the target concentration and slightly below the internal specification limit of 90%. Therefore, the actual treatment was done at a dose level of 88 mg/kg bw/day. The homogeneity of the samples was also verified for the low and high dose groups using samples from the bottom, middle, and top of the beaker.

The stability of test substance in 1 % carboxymethylcellulose suspension in drinking water was demonstrated using ion chromatography with a Metrohm Metrosep C4, 250 column. Based on the peak area measurements from 0 - 8 days, the compound was found to be stable for a period of 8 days at a refrigerated temperature. Generally the concentrations of AGBC in 1% carboxymethylcellulose suspension in drinking water were found to be in the range 90% - 110% of the nominal concentration.

Duration of treatment / exposure:

Males were exposed for 28 days: 2 weeks prior to mating, during mating and up to the day prior to scheduled necrospy. Females were exposed for up to 55 days: 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 88, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a Repeated Dose 28 Day Oral Toxicity in Rodents study conducted according to OECD guideline 407.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality/viability at least twice daily on working days or once daily on weekends and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Cage side examination was conducted at least once a day for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on gestation day 0, 7, 14 and 20. Females with litters were weighed on the day of parturition (PND 0) and PND 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly for male and female parental animals with the following exceptions: Food consumption was not determined after the 2nd week premating and during the mating period. Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20. Food consumption of parental animals which gave birth to a litter was determined on PND 1-4.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Oestrous cyclicity (parental animals):

The numbers of former implantation sites and corpora lutea were recorded for all paired females

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis and epididymis weight

Litter observations:

Each litter was examined to determine the following, if practically possible:
-Mortality/viability: The numbers of live dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
-Clinical Signs - At least once daily , detailed clinical observations were conducted for all animals.
-Bodyweights: Live pups were weighed on Days 1 and 4 of lactation
-Sex - Sex was determined for all pups on Days 1 and 4 of lactation

Postmortem examinations (parental animals):

SACRIFICE
All prenatal animals were sacrificed by decapitation under isoflurane anaesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology, with special attention to reproductive organs including epididymides, testes, ovaries and uterus.

ORGAN WEIGHTS
The following weights were determined for all animals sacrificed on schedule:
Males: animal weight, epididymides, testes
Femals: aniamal weight, adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, thymus, thyroid gland, uterus with cervix

HISTOPATHOLOGY

Samples of the following tissues and organs were collected and fixed with 4% neutral-buffered formaldehyde solution
Males: all gross lesions, coagulating glands, epididymides (modified Davidson's solution), prostate gland, seminal vesicles, testes (modified Davdson's solution)
Females: adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, colon, duodenum, esophagus, extraorbital lacrimal glands, eyes (with optic nerve (modified Davidson's solution), femur including knee joint, harderian glands, heart, ileum, jejunum (with Peyer's patches), kidneys, larynx, liver, lung, lymph nodes (mesenteric and axillary), mammary gland, nose (nasal cavity), ovaries (modified Davidson's solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, rectum, salivary glands (mandibular, sublingual), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, horacic, lumbar), spleen, sternum with bone marrow, stomach (forestomach and glandular stomach), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina and all gross lesions.

The organs were trimmed according to the "Revised guide for Organs sampling and trimming in rats and mice."

All samples from the following groups were examined by light microscopy and assessment of the findings was provided:
Testes: males, control and high dose groups
Epididymides: males, control and high dose groups
Ovaries: females, all dose groups
Brain: females, control and high dose groups
Kidneys: females, all dose groups
Liver: females, all dose groups
Spinal cord: females, control and high dose groups
Sciatic nerve: females, control and high dose groups
Thymus: females, all dose groups

A peer review on the histopathology data was performed by a second pathologist including ovaries, brain, spinal cord and sciatic nerve.

Postmortem examinations (offspring):

On PND 4, all surviving pups were sacrificed under Isoflurane anaesthesia with CO2. All pups with findings in the great vessels and 5 control pups were preserved in 4% neutral buffered formaldehyde after gross examination for histotechnical processing and histopathological examination

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnet- test (2-sided) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Fisher Exact test or the Wilcoxon test with the Bonferoni – Holm adjustment was applied to frequency data.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Food consumption (parental animals), body weight, body weight change, and gestation days were analyzed using the Dunnett test.

Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index, females with stillborn pups, and females with all stillborn pups were analyzed with the Fisher's Exact test.

Mating days until day 0 postconception, % postimplantation loss, pups stillborn, and % perinatal loss were analyzed with Wilcoxon test (one-sided +) with the Bonferroni-Holm adjustment.

Implantation sites, pups delivered, pups liveborn, live pups day x, and viability index were analyzed using the Wilcoxon test (one-sided -) with the Bonferroni-Holm adjustment.

% live male day x and % live female day x were analyzed with the Wilcoxon test (two-sided).

Weight parameters were analyzed with the Kruskal-Wallis test (two-sided).

Reproductive indices:

For each group, the following calculations were performed:
- Male Mating index (%) = (Number of males with confirmed mating* / Number of males placed with females) x 100
- Male Fertility index (%) = (Number of males proving their fertility** / Number of males placed with females) x 100
- Female Mating index (%) = (Number of females mated* / Number of females paired with males) x 100
- Female Fertility index (%) = (Number of pregnant females** / Number of females mated*) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females**) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
* - defined as female(s) with vaginal sperm or implants in utero
**- defined as female(s) with implants in utero

Offspring viability indices:

For each group, the following calculations were performed:
-Live birth index (%) = (number of live born pups at birth / total number of pups born) x 100
-Postimplantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) x 100
-Viability index (%) = Number of live pups on day 4 after birth / number of live pups on the day of birth x 100
-Sex ratio = (Number of live male OR female pups on day 0 or 4 / number of live male AND female pups on day 0 or 4) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

high dose females: paresis of hindlimbs, unsteady gait

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose females and males were statistically significantly below the concurrent control values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

High dose females and males were statistically significantly below the concurrent control values.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related findings were observed for the liver, kidneys, ovaries, and thymus of female animals, particularly in the high dose group.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Intrauterine loss and number of stillborn pups were statistically significantly higher in the high dose group.

MORTALITY
One female at 1000 mg/kg was sacrificed moribund on PND 12 because it showed piloerection, high stepping gait, paresis of both hind limbs, unsteady gate, hypothermia, poor general condition and urine stained anogenital region. This was assessed as treatment related.
CLINICAL SIGNS
In total, seven high dose females displayed clinical signs of toxicity throughout the gestation and lactation period including paresis of hind limbs, high stepping gait and piloerection.
Several male rats in the high dose group and females from both the mid and high dose group showed salivation after treatment during premating, gestation and lactation. This effect was observed immediately after treatment and was likely caused by the unpleasant taste of the test substance or by local irritation to the upper digestive tract. It is not considered to be a sign of systemic toxicity.
No clinical signs of toxicity, which may be attributed to the test substance, were detected in any male or female F0 generation parental animal of the mid and low dose group during the entire study including the gestation and lactation periods.
BODYWEIGHTS
Mean body weights of the high dose F0 males were significantly below the control animals on premating day 13 (~5%) and on mating day 8 (~6%) A similar effect was also seen in body weight change of the high dose F0 males. However, this effect was considered to be spontaneous in nature and not treatment-related.
Mean body weights of high dose females were statistically significantly below control values on GD 20 (~8%). Furthermore, the body weight change of high dose F0 females was significantly lower than controls during the gestation period.
FOOD CONSUMPTION
Food consumption of high dose F0 males and females was statistically significantly below the control group during premating, approximately 19% and 17%, respectively.
ORGAN WEIGHTS
Target organs in the F0 generation parental animals were liver, kidneys, ovaries and thymus. The absolute (+11%) and relative (+19%) liver weights were significantly increased in females of the high dose group. The increased liver weights correlated with a minimal diffuse hepatocellular hypertrophy that was observed in 7/10 animals in the high dose group. These findings were considered to be treatment related.
The absolute and relative thymus weights were dose relatedly decreased in females of the mid and high dose group.
MICROSCOPIC EXAMINATION
Liver
Minimal diffuse hepatocellular hypertrophy was observed in 7/10 animals in the high dose group. These findings were considered to be treatment related and adaptive.
Thyroid glands
The decreased thymus weights correlate with a slightly increased incidence of reduced cellularity (minimal to severe) in the cortex that was noted in 4/10 females in the high dose group in comparison to one affected control female. There was no histopathological correlation to the decrease in thymus weights in females of the mid dose group. All these findings were considered to be treatment related and adverse.
Kidneys
Slight chronic nephropathy was observed in the kidneys of 7/10 females in the high dose group. This finding was mainly located in the cortex and occurred in 2 females bilaterally and was unilateral in 5 females. One control female showed minimal chronic nephropathy.
5/10 animals of the high dose group showed multifocal hypertrophy of the proximal tubular cells in the outer stripe of outer medulla (OSOM). This finding correlates with the increased absolute and relative kidney weights in the test group.
Ovaries
One control animal, one female of the mid dose group and 4 of the high dose group showed an increase in the number of atretic follicles.
REPRODUCTIVE PARAMETERS
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
DEVELOPMENTAL DATA
The test substance adversely affected the pre-and postnatal development off offspring at 1000 mg/kg bw/day. This was evident by increased pre-and post-natal mortality and reduced pup body weights shortly after birth. Furthermore, a significantly high proportion of pups, across all treatment groups (36 /59 live born pups) had microscopic and/or macroscopic evidence of dissecting aneurysms.
Gestation
The mean duration of gestation varied between 22.8 (high dose group), 22.0 (mid and low dose group) and 22.1 (control) days. As the high dose value is outside the historical control range, the prolonged duration of gestation in this dose group is considered to be treatment related.

Dose descriptor:

NOAEL

Remarks:

General Systemic Toxicity

Effect level:

ca. 300 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Decreased food consumption and body weight/body weight changes were seen at 1000 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

General Systemic Toxicity

Effect level:

88 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Decreased absolute and relative thymus weights at 300 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

Fertility and Reproductive Performance

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No effects at highest dose tested.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The pup viability in the high dose group was significantly reduced.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Female and male pups weights in the high dose group were statistically decreased.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pups in all dose groups experienced effects in hte pericardial vessels including descending aorta and aneurysms.

VIABILITY (OFFSPRING)
The mean number of live born pups was statistically significantly lower in the high dose groups. Furthermore, the number of litters with still born pups was statistically increased on the high dose group. 2 females of the high dose group had complete litter losses on day 1 and 2 PND, respectively.
The viability index indicating pup mortality during lactation (PND 0-4) varied between 100%/ 98.8% / 100% and 67.6% in control, low, mid and high dose groups respectively. The reduced viability index in the high dose group is considered to be treatment related.
CLINICAL SIGNS (OFFSPRING)
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
One male pup of the high dose group showed a spontaneous finding (short tail) which was not considered to be treatment related.
BODY WEIGHT (OFFSPRING)
Mean body weights of high dose F1 female pups were statistically significantly below control values on PND (~21%) In F1 males of this dose group mean body weights were also lower than controls (~11%). Although not statistically significant, body weight impairment was considered a direct effect of the test substance, On PND 4 the mean body weights of all pups of the high dose group were still below the control data.
One male and five female runts were seen in test group 3 and one male runt was seen in the control group
OTHER FINDINGS (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance treatment group. Any slight differences were regarded as spontaneous in nature.
NECROSPY (OFFSPRING)
Macroscopic examination of pups at necrospsy revealed a number of findings concerning the pericardial vessel in all dose groups, which are considered to be substance-induced. Necrospy observation across all dose groups (excluding control) included dilated ductus arteriosus, aneurysm, descending aorta, dilated aorta, dilated pulmonary artery, dilated ductus arteriosus, dilated aortic arch and dilated pulmonary trunk. 16 male pups in the high dose group, 3 in the mid dose group and 2 in the low dose group showed dilation in great vessel macroscopically compared to 22 females in the high dose group and 1 in the mid dose group. 15 male pups in the high dose group, 3 in the mid dose group and 1 in the low dose had aneurysms histopathologically compared with 22 high dose females, and 1 mid dose female.

Dose descriptor:

LOAEL

Remarks:

Developmental Toxicity

Generation:

F1

Effect level:

88 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Increased pre- and postnatal mortality and reduced pup body weights were evident only at the 1000 mg/kg bw/day dose level. However, dissecting aneurysms were detected in a dose dependent fashion beginning at the lowest dose level of 88 mg/kg bw/day.

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the present OECD 421 reproduction/developmental toxicity screening study in Wistar rats, the following NOAELs of the test substance can be determined:
The NOAEL for generic systemic toxicity for males was 300 mg/kg bw/day based on decreased food consumption and body weighs/ body weight changes at 1000 mg/kg bw/day. The NOAEL for females was 88 mg/kg bw/day as they showed decreased absolute and relative thymus weights at 300 mg/kg bw/day.
The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day as no effects were seen on these parameters.
The NOAEL for developmental toxicity in the F1 offspring was not determined as dissecting aneurysms were detected in a dose dependent fashion across all treatment groups.

Executive summary:

A reproduction/ developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 421 and EPA OPPTS 870.3550 under GLP conditions.

Four groups of ten male and female Wistar Han rats were exposed by oral gavage to the test material at 0, 88, 300 and 1000 mg/kg bw/day in carboxymethylcellulose. Males were exposed for 28 days ( 2 weeks prior to mating, during mating, and up to termination) and females were exposed for up to 55 days ( 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation).

Animals were evaluated for mortality/ viability, clinical and functional observations, body weights and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

Severe clinical findings were observed in several females at 1000 mg/kg throughout the gestation and lactation period including paresis of hindlimbs, high stepping gait and piloerection. No clinical signs directly related to the test item were observed in any other group. The mating, fertility and conception indices, precoital time and number of corpora lutea and implantation sites were unaffected by treatment. However, the test substance adversely affected the pre-and post-natal development off spring at 1000 mg/kg bw/day which was evident by increased pre and post- natal mortality and reduced pup weights. Furthermore, a significantly high proportion of pups, across all treatment groups had microscopic and/or macroscopic evidence of dissecting aneurysms. The mean duration of gestation varied between 22.0 and 22.8 across all dose groups. As the high dose value (22.8 days) is outside the historical control range, the prolonged duration of gestation in this dose group is considered to be treatment related.

Under the conditions of this study, the NOAEL for general systemic toxicity for males was 300 mg/kg bw/day based on decreased food consumption and body weights/ body weight change at 1000 mg/kg bw/day. For females the NOAEL was 88 mg/kg bw/day as they showed decreased absolute and relative thymus weights at 300 mg/kg bw/day in addition. 

The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day as no effects were seen on these parameters.

The NOAEL for developmental toxicity in the F1 offspring was not determined, and a LOAEL of 88 mg/kg bw/day was calculated. Although increased pre-and post-natal mortality and reduced pup weights were evident only at 1000 mg/kg bw/day dose level, dissecting aneurysms were detected in a dose-dependent fashion beginning at the lowest dose level of 88 mg/kg bw/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was conducted in accordance with the standardised guideline OECD 421 under GLP conditions. It was assigned a reliablity score of 1 in accordance with the criteria set forth by Klimisch (1997).

Additional information

A reproduction/ developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 421 and EPA OPPTS 870.3550 under GLP conditions.

Four groups of ten male and female Wistar Han rats were exposed by oral gavage to the test material at 0, 88, 300 and 1000 mg/kg bw/day in carboxymethylcellulose. Males were exposed for 28 days ( 2 weeks prior to mating, during mating, and up to termination) and females were exposed for up to 55 days ( 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation).

Animals were evaluated for mortality/ viability, clinical and functional observations, body weights and food consumption, clinical pathology, macroscopic evaluation at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

Severe clinical findings were observed in several females at 1000 mg/kg throughout the gestation and lactation period including paresis of hindlimbs, high stepping gait and piloerection. No clinical signs directly related to the test item were observed in any other group. The mating, fertility and conception indices, precoital time and number of corpora lutea and implantation sites were unaffected by treatment. The mean duration of gestation varied between 22.0 and 22.8 across all dose groups. 

Under the conditions of this study, the NOAEL for general systemic toxicity was 300 mg/kg bw/day in males based on decreased food consumption and body weights/ body weight change at 1000 mg/kg bw/day. For females the NOAEL was 88 mg/kg bw/day as they showed decreased absolute and relative thymus weights at 300 mg/kg bw/day. 

The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day as no effects were seen on these parameters.

Short description of key information:
No adverese effects on fertility or mating were observed at the highest tested group.

Justification for selection of Effect on fertility via oral route:
Only one study available

Effects on developmental toxicity

Description of key information

Increased pre and post-natal mortality and reduced pup body weights were evident only at 1000 mg/kg bw/day.  In addition, dissecting aneurysms were detected in a dose dependent fashion beginning at the lowest dose level of 88 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2014 to 16 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Strain: Crl:WI(Han) .
- Age at study initiation: 10-12 weeks
- Weight at study initiation: The average weight of the males was between 311.0 g and 361.5 g and the average weight of the females was between 165.5 g and 220 g on Day 1 of the study.
- Housing: Rats were held individually in Polycarbonate type M III cages (floor area of about 800 cm2), with the following exceptions: During overnight mating's, male and female mating partners were housed together in Polycarbonate type M III cages and pregnant animals and their litters were housed together until PND 4.
- Diet (e.g. ad libitum): Free access to ground Kliba maintenance diet mouse/rat 'GLP' meal
- Water (e.g. ad libitum): Water Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C
- Humidity (%): 30 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in drinking water

Details on exposure:

VEHICLE
-Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
-Dose Volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Method of Formulation – The test substance suspension was weighed in a calibrated beaker depending on the dose group, topped with 1% carboymethylcellulose suspension in drinking water and intensely mixed with a homogenizer. During administration, the preparations were kept homogenous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The average concentration of AGBC in 1% carboxymethylcellulose suspension in drinking water for the three dosing groups was measured using potentiometric tiration with trifluoromethanesulfonic acid. The middle and high dose groups had a concentration of 96% of the expected dose. In the low dose group, the measured concentration was 88% of the target concentration and slightly below the internal specification limit of 90%. Therefore, the actual treatment was done at a dose level of 88 mg/kg bw/day. The homogeneity of the samples was also verified for the low and high dose groups using samples from the bottom, middle, and top of the beaker.

The stability of test substance in 1 % carboxymethylcellulose suspension in drinking water was demonstrated using ion chromatography with a Metrohm Metrosep C4, 250 column. Based on the peak area measurements from 0 - 8 days, the compound was found to be stable for a period of 8 days at a refrigerated temperature. Generally the concentrations of AGBC in 1% carboxymethylcellulose suspension in drinking water were found to be in the range 90% - 110% of the nominal concentration.

Details on mating procedure:

Following a minimum of 14 days of exposure for males and females, one female was cohabitated with one male of the same treatment group. Detection of mating was confirmed by evidence of sperm in the vaginal smear. This day was designated DAY 0 post-coitum. Male and female rats were separated and housed individually after mating was confirmed. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Duration of treatment / exposure:

Males were exposed for 28 days: 2 weeks prior to mating and up to the day prior to scheduled necrospy. Females were exposed for up to 55 days: 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 88, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a Repeated Dose 28 day Oral Toxicity Study in Rodents study conducted according to OECD guideline 407.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality/viability at least twice daily on working days or once daily on weekends and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Cage side examination was conducted at least once a day for any signs of morbidity, pertinent behavioural changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on gestation day 0, 7, 14 and 20. Females with litters were weighed on the day of parturition (PND 0) and PND 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly for male and female parental animals with the following exceptions: Food consumption was not determined after the 2nd week premating and during the mating period. Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20. Food consumption of parental animals which gave birth to a litter was determined on PND 1-4.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Fetal examinations:

Each litter was examined to determine the following, if practically possible:
-Mortality/viability: The numbers of live dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
-Clinical Signs: At least once daily , detailed clinical observations were conducted for all animals.
-Bodyweights: Live pups were weighed on Days 1 and 4 of lactation
-Sex: Sex was determined for all pups on Days 1 and 4 of lactation

Statistics:

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnet- test (2-sided) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Fisher Exact test or the Wilcoxon test with the Bonferoni – Holm adjustment was applied to frequency data.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Food consumption (parental animals), body weight, body weight change, and gestation days were analyzed using the Dunnett test.

Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index, females with stillborn pups, and females with all stillborn pups were analyzed with the Fisher's Exact test.

Mating days until day 0 postconception, % postimplantation loss, pups stillborn, and % perinatal loss were analyzed with Wilcoxon test (one-sided +) with the Bonferroni-Holm adjustment.

Implantation sites, pups delivered, pups liveborn, live pups day x, and viability index were analyzed using the Wilcoxon test (one-sided -) with the Bonferroni-Holm adjustment.

% live male day x and % live female day x were analyzed with the Wilcoxon test (two-sided).

Weight parameters were analyzed with the Kruskal-Wallis test (two-sided).

Indices:

For each group, the following calculations were performed:
- Male Mating index (%) = (Number of males with confirmed mating* / Number of males placed with females) x 100
- Male Fertility index (%) = (Number of males proving their fertility** / Number of males placed with females) x 100
- Female Mating index (%) = (Number of females mated* / Number of females paired with males) x 100
- Female Fertility index (%) = (Number of pregnant females** / Number of females mated*) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females**) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
-Live birth index (%) = (number of live born pups at birth / total number of pups born) x 100
-Postimplantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) x 100
-Viability index (%) = Number of live pups on day 4 after birth / number of live pups on the day of birth x 100
-Sex ratio = (Number of live male OR female pups on day 0 or 4 / number of live male AND female pups on day 0 or 4) x 100

* - defined as female(s) with vaginal sperm or implants in utero
**- defined as female(s) with implants in utero

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Increased gestation length in high dose females

Details on maternal toxic effects:
MORTALITY
One female at 1000 mg/kg was sacrificed moribund on PND 12 because it showed piloerection, high stepping gait, paresis of both hind limbs, unsteady gate, hypothermia, poor general condition and urine stained anogenital region. This was assessed as treatment related.
CLINICAL SIGNS
In total, seven high dose females displayed clinical signs of toxicity throughout the gestation and lactation period including paresis of hind limbs, high stepping gait and piloerection.
Several male rats in the high dose group and females from both the mid and high dose group showed salivation after treatment during premating, gestation and lactation. This effect was observed immediately after treatment and was likely caused by the unpleasant taste of the test substance or by local irritation to the upper digestive tract. It is not considered to be a sign of systemic toxicity.
No clinical signs of toxicity, which may be attributed to the test substance, were detected in any male or female F0 generation parental animal of the mid and low dose group during the entire study including the gestation and lactation periods.
BODYWEIGHTS
Mean body weights of the high dose F0 males were significantly below the control animals on premating day 13 (~5%) and on mating day 8 (~6%) A similar effect was also seen in body weight change of the high dose F0 males. However, this effect was considered to be spontaneous in nature and not treatment-related.
Mean body weights of high dose females were statistically significantly below control values on GD 20 (~8%). Furthermore, the body weight change of high dose F0 females was significantly lower than controls during the gestation period.
FOOD CONSUMPTION
Food consumption of high dose F0 males and females was statistically significantly below the control group during premating, approximately 19% and 17%, respectively.
ORGAN WEIGHTS
Target organs in the F0 generation parental animals were liver, kidneys, ovaries and thymus. The absolute (+11%) and relative (+19%) liver weights were significantly increased in females of the high dose group. The increased liver weights correlated with a minimal diffuse hepatocellular hypertrophy that was observed in 7/10 animals in the high dose group. These findings were considered to be treatment related.
The absolute and relative thymus weights were dose relatedly decreased in females of the mid and high dose group.
MICROSCOPIC EXAMINATION
Liver
Minimal diffuse hepatocellular hypertrophy was observed in 7/10 animals in the high dose group. These findings were considered to be treatment related and adaptive
Thyroid glands
The decreased thymus weights correlate with a slightly increased incidence of reduced cellularity (minimal to severe) in the cortex that was noted in 4/10 females in the high dose group in comparison to one affected control female. There was no histopathological correlation to the decrease in thymus weights in females of the mid dose group. All these findings were considered to be treatment related and adverse
Kidneys
Slight chronic nephropathy was observed in the kidneys of 7/10 females in the high dose group. This finding was mainly located in the cortex and occurred in 2 females bilaterally and was unilateral in 5 females. One control female showed minimal chronic nephropathy.
5/10 animals of the high dose group showed multifocal hypertrophy of the proximal tubular cells in the outer stripe of outer medulla (OSOM). This finding correlates with the increased absolute and relative kidney weights in the test group.
Ovaries
One control animal, one female of the mid dose group and 4 of the high dose group showed an increase in the number of atretic follicles.
REPRODUCTIVE PARAMETERS
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
DEVELOPMENTAL DATA
The test substance adversely affected the pre-and postnatal development off offspring at 1000 mg/kg bw/day. This was evident by increased pre-and post-natal mortality and reduced pup body weights shortly after birth. Furthermore, a significantly high proportion of pups, across all treatment groups (36 /59 live born pups) had microscopic and/or macroscopic evidence of dissecting aneurysms.
Gestation
The mean duration of gestation varied between 22.8 (high dose group), 22.0 (mid and low dose group) and 22.1 (control) days. As the high dose value is outside the historical control range, the prolonged duration of gestation in this dose group is considered to be treatment related.

Dose descriptor:

LOAEL

Effect level:

88 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Dissecting aneurysms were evident in all pups across all treatment groups.

Details on embryotoxic / teratogenic effects:
DEVELOPMENTAL DATA
The test substance adversely affected the pre-and postnatal development off offspring at 1000 mg/kg bw/day. This was evident by increased pre-and post-natal mortality and reduced pup body weights shortly after birth. Furthermore, a significantly high proportion of pups, across all treatment groups (36 /59 live born pups) had microscopic and/or macroscopic evidence of dissecting aneurysms.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of the present OECD 421 reproduction/developmental toxicity screening study in Wistar rats, the following NOAELs of the test substance can be determined:
The NOAEL for generic systemic toxicity for males was 300 mg/kg bw/day based on decreased food consumption and body weighs/ body weight changes at 1000 mg/kg bw/day. The NOAEL for females was 88 mg/kg bw/day as they showed decreased absolute and relative thymus weights at 300 mg/kg bw/day.
The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day as no effects were seen on these parameters.
The NOAEL for developmental toxicity in the F1 offspring was not determined as dissecting aneurysms were detected in a dose dependent fashion across all treatment groups.

Executive summary:

A reproduction/ developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 421 and EPA OPPTS 870.3550 under CLP conditions.

Four groups of ten male and female Wistar Han rats were exposed by oral gavage to the test material at 0, 88, 300 and 1000 mg/kg bw/day in carboxymethylcellulose. Males were exposed for 28 days ( 2 weeks prior to mating, during mating, and up to termination) and females were exposed for up to 55 days ( 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation).

Animals were evaluated for mortality/ viability, clinical and functional observations, body weights and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

Severe clinical findings were observed in several females at 1000 mg/kg throughout the gestation and lactation period including paresis of hindlimbs, high stepping gait and piloerection. No clinical signs directly related to the test item were observed in any other group. The mating, fertility and conception indices, precoital time and number of corpora lutea and implantation sites were unaffected by treatment. However, the test substance adversely affected the pre-and post-natal development off spring at 1000 mg/kg bw/day which was evident by increased pre and post- natal mortality and reduced pup weights. Furthermore, a significantly high proportion of pups, across all treatment groups had microscopic and/or macroscopic evidence of dissecting aneurysms. The mean duration of gestation varied between 22.0 and 22.8 across all dose groups. As the high dose value (22.8 days) is outside the historical control range, the prolonged duration of gestation in this dose group is considered to be treatment related.

Under the conditions of this study, the NOAEL for general systemic toxicity for males was 300 mg/kg bw/day based on decreased food consumption and body weights/ body weight change at 1000 mg/kg bw/day. For females the NOAEL was 88 mg/kg bw/day as they showed decreased absolute and relative thymus weights at 300 mg/kg bw/day in addition. 

The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day as no effects were seen on these parameters.

The NOAEL for developmental toxicity in the F1 offspring was not determined, and a LOAEL of 88 mg/kg bw/day was calculated. Although increased pre-and post-natal mortality and reduced pup weights were evident only at 1000 mg/kg bw/day dose level, dissecting aneurysms were detected in a dose-dependent fashion beginning at the lowest dose level of 88 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

88 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

the key study was conducted in accordance with the standardised guideline OECD 421 under GLP conditions. It wa assigned a reliability score of 1 in accordance with the criteria set forth by klimisch (1997).

Additional information

A reproduction/ developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 421 and EPA OPPTS 870.3550 under CLP conditions.

Four groups of ten male and female Wistar Han rats were exposed by oral gavage to the test material at 0, 88, 300 and 1000 mg/kg bw/day in carboxymethylcellulose. Males were exposed for 28 days ( 2 weeks prior to mating, during mating, and up to termination) and females were exposed for up to 55 days ( 2 weeks prior to mating, during mating and gestation and during at least 4 days of lactation).

Animals were evaluated for mortality/ viability, clinical and functional observations, body weights and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

The test substance adversely affected the pre-and post-natal development offspring at 1000 mg/kg bw/day which was evident by increased pre and post- natal mortality and reduced pup weights. Furthermore, a significantly high proportion of pups, across all treatment groups had microscopic and/or macroscopic evidence of dissecting aneurysms. The mean duration of gestation varied between 22.0 and 22.8 across all dose groups. As the high dose value (22.8 days) is outside the historical control range, the prolonged duration of gestation in this dose group is considered to be treatment related.

The NOAEL for developmental toxicity in the F1 offspring was not determined, and a LOAEL of 88 mg/kg bw/day was calculated. Although increased pre-and post-natal mortality and reduced pup weights were evident only at 1000 mg/kg bw/day dose level, dissecting aneurysms were detected in a dose-dependent fashion beginning at the lowest dose level of 88 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one reliable key study available

Justification for classification or non-classification

AGBC will be classified as a Reproductive Toxicant Category 1B under CLP based on the adverse effects seen in a Klimisch rated 1, OECD compliant, 421 reproductive/developmental screening test.