Tetramethylammonium hydrogen phthalate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17JAN2014-12MAR2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) (July 1995)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen) (July 2000)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetramethylammonium hydrogen phthalate

EC Number:

416-900-5

EC Name:

Tetramethylammonium hydrogen phthalate

Cas Number:

79723-02-7

Molecular formula:

C12H17NO4

IUPAC Name:

tetramethylazanium 2-carboxybenzoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report):TMAHP
- Cas nr.: 79723-02-7
- Substance type: organic
- Physical state: powder
- Expiration date of the lot/batch: September/October 2016
- Storage condition of test material: At room temperature in the dark in well-sealed container
- pH: 5-10 for solution

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
- Age at study initiation: Approx. 10-11 weeks
- Weight at study initiation: Males: 302-342 g; Females: 186-219 g
- Housing: 5 animals/sex/cage in Macrolon plastic cages (pre-mating, post-mating males), or individually (post-mating females)
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18- 24
- Humidity (%): 40-70
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17JAN2014 To: 12MAR2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No correction was made for the purity/composition of the test substance.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg body weight
JUSTIFICATION OF VEHICLE:
- Based on trial formulations performed at WIL Research Europe and based on information provided by the sponsor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion after the treatment phase on samples as specified in the protocol, according to a validated method (WIL Project 504504 and 500814). These formulations were prepared in the same manner as used during the study. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
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%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. 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, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 – 46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week

Duration of test:

Maternal animals and pups were sacrificed 4 days post partum.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 15, 35 (75 up to day 6) mg/ kg bw/ day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

One female from control group, 3 from the group dosed at 15 mg/kg bw/ d and 2 from group dosed at 35 mg/kg bw/ d were not dosed on day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Dose levels were based on the results of a previous 28-Day study (SafePharm Laboratories, Project 13/553) where animals were exposed to TMAP at 5, 15 and 75 mg/kg bw/day. The NOAEL for both sexes was established at 15 mg/kg/day.

Examinations

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were conducted for all animals between 1 and 2 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. An extra body weight measurement was performed on Day 7, the day on which the lowered highest dose level was in effect.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Weekly, except for females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation. An extra food consumption measurement was determined on Day 7, the day on which the lowered highest dose level was in effect.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- 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 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 and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: 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.
- All pups were killed by decapitation between days 5-7 of lactation. At necropsy, all pups were sexed by examination of the internal sex organs and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- 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.
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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Indices:

Reproductive indices
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100;
- Fertility index: Number of pregnant females/Number of females paired 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.

Offspring viability indices
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100;
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100;
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100;
- Viability index: Number of live pups before planned necropsy/ Number of pups born alive x 100.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment with 75 mg/kg produced severe toxicity including mortality of 3 animals and significantly lower body weights, enlarged livers and decreased food consumption. After the dose level was lowered to 35 mg/kg, males had lower body weights through the duration of treatment, though some recovery was seen. Females reached control levels during the post coitum and lactation period. Females at 35 mg/kg had higher liver weights than controls. Microscopically, this corresponded to an increased amount of glycogen in the liver for these females. This was determined to be treatment related but was not considered adverse. Taken together, the effects seen at 35 mg/kg were not considered to be adverse. One female dosed at 35 mg/kg bw/day had a total litter loss. Her macroscopic findings included pale discoloration of the liver and reduced size and tan discoloration of the thymus.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

35 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 35 mg/kg bw/day (actual dose received)

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:
GESTATION
The gestation index and duration of gestation were unaffected with treatment up to 35 mg/kg. There were 9, 10, 10 and 9 pregnant females in the control, 5, 15 and 35 mg/kg groups, respectively.

PARTURITION/ MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT
The number of living and dead pups at first litter check and the sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. Mean litter sizes were 12.3, 12.3, 9.5 and 11.0 pups/litter in the control, 5, 15 and 35 mg/kg groups,
respectively.

MORTALITY
No pups of the control group, one pup each in the 5 and 15 mg/kg groups and 13 pups at 35 mg/kg were found dead, killed in extremis or went missing during the first days of lactation. Missing pups were most likely cannibalised. With 13 dead/missing pups, females at 35 mg/kg had significantly higher pup mortality and a correspondingly lower viability index compared to controls. This was entirely attributable to one female who lost her entire litter of 13 pups by Day 2. There were no other dead or missing pups in this treatment group apart from this litter. It is of note that the liver of the maternal rat of this litter was found to have a discoulored and pale liver and a thymus that was reduced in size and discoulored as well (tan colour). Consequently, the higher pup mortality was not considered to reflect treatment-related developmental toxicity. No toxicological relevance was attributed to the individual dead/missing pups in the other treatment groups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS
Incidental clinical symptoms of pups consisted of small size, lean appearance, cold, blue nose, scabs on the nose or hind leg, missing toe and blue paw. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

BODY WEIGHTS
There were no adverse effects on pup body weights up to 35 mg/kg. At 15 mg/kg, male pup body weights (and combined weights) were significantly higher than controls on post natal Day 4. This was secondary to relatively high body weights of pups in smaller litters (2 litters) and was not considered treatment related or toxicologically relevant.

MACROSCOPY
Incidental clinical symptoms of pups consisted of small size, lean appearance, cold, blue nose, scabs on the nose or hind leg, missing toe and blue paw. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis was conducted on two compounds: Phthalic acid and TMA. The concentrations analysed in the formulations of all groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and homogeneous (i.e. coefficient of variation ≤ 10%). No test substance was detected in the control formulation. Only the concentrations analysed in the formulations of the highest dose group based on the tetramethylammonium part of the test substance were above the target concentration (i.e. mean accuracy of 124%). The high mean accuracy is probably caused by the increasing sensitivity during the analytical measurement sequence. This was also observed for the procedural recovery samples indicating that the analyzed concentrations are overestimated. Besides this, the concentrations analyzed based on the phthalic acid part of the test substance were very close to the nominal concentrations, indicating that the formulations were prepared properly. Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:

In a reproduction/developmental toxicity screening test of TMAHP in rats by oral gavage performed according to OECD guideline 421 and according to GLP principles, a parental and developmental No Observed Adverse Effect Level (NOAEL) of 35 mg/kg bw/ day was established. The LOAEL parental was found to be 75 mg/kg bw/day.

Executive summary:

A reproduction/developmental toxicity screening test was conducted according to OECD guideline 421 and according to GLP principles with TMAHP. Rats were exposed to the test substance by oral gavage at dose levels of 5, 15 35 and 75 mg/kg. Based on mortality in the highest dose group in the first week (2 males and 1 female), lower body weight gains or weight loss compared to controls on Day 7 of the treatment period (coinciding with reduced food intake), the highest dose was lowered to 35 mg/kg bw/day after the first week. No further mortality occurred. No toxicologically relevant differences in body weight gain of treated groups compared to controls were noted. Females at 35 mg/kg had significantly higher liver weights than controls (25%). Microscopically, this corresponded to an increased amount of glycogen in the liver for these females. This was determined to be treatment related but was not considered adverse. No treatment-related changes were noted in the clinical appearance or macroscopic findings. No toxicologically relevant effects on the gestation index and duration, parturition, maternal care or early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Based on these data, the parental and developmental No Observed Adverse Effect Level (NOAEL) was found to be 35 mg/kg bw/day.