1-Propanaminium, N-(carboxymethyl)-3-(formylamino)-N,N-dimethyl-, inner salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2011 - 26 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

PC-2011-350

IUPAC Name:

PC-2011-350

Test material form:

other: Clear colourless to yellowish liquid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: males and females. Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: mean weight of females range at start of post-coitum was 215 gram.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-coitum: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days
- Identification: Animals were only identified by tattoo on the foot and not by earmark as well. Animals were individually housed with unique identification, thus this has no impact on the study’s integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 21.5°C
- Humidity (%): 42 - 89%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 20 December 2011 - 26 January 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Corrections were made
for the purity and the density of the test substance.

Storage conditions of formulations:
At ambient temperature.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (06 January 2012), according to a validated method (NOTOX Project 454117). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and no test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male ).
- Age at mating of the mated females in the study: Approximately 12 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Duration of treatment / exposure:

Females were exposed from Days 6 to 19 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Duration of treatment: From Days 6 to 19 post-coitum, inclusive.

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of the pilot study (NOTOX Project 497618 (see attached document), based on the results from the 14-day pilot to the 90-day study (NOTOX Project 497923) and the results of the 90-day study (NOTOX Project 497622) available at initiation of the
main prenatal developmental toxicity study.
- Rationale for animal assignment: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from Day 0 post-coitum onwards. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

NEUROBEHAVIOURAL EXAMINATION:
No

BODY WEIGHT:
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum

FOOD CONSUMPTION:
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION :
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: first 10 pregnant females/group. Instead of 10 animals, blood was taken from 14, 13, 12 and 13 animals from Groups 1, 2, 3, and 4, respectively. The additional data is reported and has no adverse impact on the study’s integrity.
- Parameters checked were: According to test guidelines


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: No the animals were not deprived of food overnight. Water was provided.
- How many animals: first 10 pregnant females/group. Instead of 10 animals, blood was taken from 13, 12, 12 and 13 animals from Groups 1, 2, 3, and 4, respectively. The additional data is reported and has no adverse impact on the study’s integrity.
- Parameters checked were: According to test guidelines

URINALYSIS:
No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses
- The number and distribution of embryo-fetal deaths
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
The uterus from one animal of Group 2 was not weighed Evaluation: Sufficient data is available for a thorough evaluation.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter of Groups 1 and 4
- Head examinations: Yes: half per litter

Statistics:

- 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 of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss. Dead fetuses, early and late resporptions and pre- and post-implantation loss were compared using the litter as the statistical unit.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected / litter (%) = (number of viable fetuses affected / litter)/(number of viable fetuses/litter) x 100

Historical control data:

report contains historical control data on malformations and variations for the rat strain used. Number of studies evaluated: 23
Total No. Fetuses/Litters Examined Externally 4557 384
Total No. Fetuses/Litters Examined Viscerally 3740 384
Total No. Fetuses/Litters Examined Skeletally 3122 376

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were 2, 1, 2 and 2 non-pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the numbers of implantation sites, corpora lutea, pre-implantation or post-implantation loss up to 1000 mg/kg. Post-implantation loss was higher at 100 mg/kg, but in the absence of a dose response it was not considered treatment related.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no effect of treatment on the litter size for any group, nor were there any effects on the numbers of viable or dead fetuses.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

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

Basis for effect level:

other: no effects up to the limit dose

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

MATERNAL FINDINGS

Mortality: There were no unscheduled deaths in the study.

Clinical signs: There were no treatment related clinical signs noted up to 1000 mg/kg. Alopecia was an incidental clinical sign noted for control and treated animals, which had no association with treatment.

Body weights: Body weights and body weight gains remained unaffected by treatment up to 1000 mg/kg.

Food consumption: There were no treatment related effects on absolute or relative food consumption up to 1000 mg/kg.

Haematology: There were no toxicologically relevant effects on haematology parameters up to 1000 mg/kg. Significant reductions in red blood cell counts (all treated groups) and in haemoglobin and haematocrit values (both at 100 and 1000 mg/kg) were seen. However, these differences were likely due to relatively high means obtained for control animals that were attributable to high values for animal nos. 1 and 3 for all three parameters (high red blood cells for one female of the control group were also seen). In the absence of treatment related effects on any other parameter, these changes were not considered to be toxicologically relevant.

Clinical biochemistry: Clinical biochemistry parameters were unaffected up to 1000 mg/kg.

Macroscopic examination: There were no macroscopic findings attributable to treatment noted up to 1000 mg/kg. Incidental findings seen for control and treated animals included alopecia on the flanks, hind legs and/or abdominal regions. Tan discoloration of the thymus was also noted for one control female and one high dose female; this high dose female also had fluid in the uterus and cervix. These findings have no toxicological relevance.

Organ weights: There were no differences in uterine weights between control and treated animals up to 1000 mg/kg.

Maternal pregnancy data: There were 2, 1, 2 and 2 non-pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the numbers of implantation sites, corpora lutea, pre-implantation or post-implantation loss up to 1000 mg/kg. Post-implantation loss was higher at 100 mg/kg, but in the absence of a dose response it was not considered treatment related.

FETAL FINDINGS

Litter size: There was no effect of treatment on the litter size for any group, nor were there any effects on the numbers of viable or dead fetuses.

Sex ratio: The sex ratio was unaffected by treatment.

Fetal body weight: There were no treatment related effects on fetal body weights up to 1000 mg/kg.

External malformations and variations: There were no test substance-related effects on fetal external morphology. Two live fetuses and a dead fetus were observed with external malformations in the mouth region. One fetus at 100 mg/kg had a small lower jaw and one fetus at 1000 mg/kg had a cleft upper lip (bilateral), cleft palate (entire length) and small upper jaw. The cleft palate and small upper jaw of one fetus 1000 mg/kg were skeletally confirmed and in addition it appeared that this fetus had fused nasal bones and fused mandibles. The dead fetus at 100 mg/kg had craniorachischisis (the cranial vault and vertebral column were open through the sacral region) and a small upper jaw. No external developmental malformations were observed in any of the other fetuses and external variations were not observed in this study. Visceral malformations and variations: There were no test substance-related effects on fetal visceral morphology. Visceral malformations were observed 0(0), 3(2), 1(1) and 1(1) fetuses (litters) in the control, 100, 300 and 1000 mg/kg groups, respectively. At 100 mg/kg, the malformations observed were small eyes in one fetus (this fetus also had a small lower jaw at external examination), retroesophageal aortic arch in one fetus and internal hydrocephaly (observed at cephalic examination) in one fetus. At 1000 mg/kg, one fetus also had internal hydrocephaly and at 300 mg/kg, one fetus only had one testis and epididymis. In addition to these fetuses with visceral malformations, the dead fetus at 100 mg/kg had a malpositioned testis.

Visceral developmental variations observed in the test substance-treated groups were partially undescended thymus horns, small supernumerary liver lobes, liver appendix, dilated ureter, pale spleen, spot on stomach, supernumerary pancreas, discolored adrenal, right subclavian originating from the aortic arch and malpositioned left carotid.

Skeletal malformations and variations

There were no test substance-related effects on fetal skeletal morphology. Skeletal malformations were observed in 3(3) fetuses (litters) in both the control and 1000 mg/kg group. At 1000 mg/kg, two fetuses had a vertebral anomaly with associated rib anomaly. The anomaly in one fetus was located in the thoracic and lumbar vertebral column region and in one fetus in the cervical and thoracic region. Also at 1000 mg/kg, one fetus which was noted with a small upper jaw and cleft lip and cleft palate externally, had fused nasal bones and fused mandibles. The malformations observed in the control group were malpositioned metatarsal in one fetus, supernumerary metatarsal in one fetus and sternoschisis in one fetus. The mean litter incidence of ossified cervical centrum no. 1 was significantly decreased at 1000 mg/kg (23.2% per litter) compared to the control group value (36.0% per litter). Other skeletal developmental variations observed more than once in the test substance-treated groups were 14th rudimentary ribs, 7th cervical rudimentary ribs, unossified sternebra nos. 5 and 6, slightly to moderately malaligned sternebrae, reduced ossification of the skull, bent ribs, reduced ossification of vertebral centra, caudal shift of pelvic girdle and 14th full ribs.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Effect Level (NOEL) for PC-2011-350 was established as being at least 1000 mg/kg body weight/day.

Executive summary:

In a developmental toxicity study according to OECD guideline 414, the testsubstance was administered to 22 female Crl:WI(Han) rats/dose by gavage at dose levels of 0, 100, 3000 and 1000 mg/kg bw/day from days 6 to 19 post-coitum.

There were no maternal treatment-related effects in mortality, clinical signs, body weight, food consumption, or cesarean parameters observed in the 100, 300 and 1000 mg/kg/day groups. The maternal NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

There were no treatment-related effects in developmental parameters observed in the 100, 300 and 1000 mg/kg/day groups. The developmental NOEL is at least 1000 mg/kg bw/day, based on no effects up to the limit dose.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rat.