Tetramethylammonium hydrogen phthalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

July 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

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 (MIV type, height 18 cm), except during mating
- 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): Approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

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

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

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.

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%.

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, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Remarks:

Doses / Concentrations:
0, 5, 15, 35 (75 up to day 6) mg/kg bw/ d
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 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.

Positive control:

No.

Parental animals: Observations and 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: Males and 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 determined on Day 7, the day on which the lowered highest dose level was in effect.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Weekly, except for males and 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.

Oestrous cyclicity (parental animals):

Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Sperm parameters (parental animals):

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Epididymides and testes weights were recorded from all F0-males on the scheduled day of necropsy.

Litter observations:

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (following completion of the mating period (a minimum of 28 days of dose administration)).
- Maternal animals: All surviving animals (females which delivered on lactation Days 5-7, females which failed to deliver and females without evidence of mating approximately 21 days after the last day of the mating period, and females with total litter loss within 24 hours of litter loss).

GROSS NECROPSY
- Gross necropsy consisted of external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the OECD guideline were prepared for microscopic examination. Liver and kidney weights and the terminal body weight were measured for all animals and epididymides and testes weights were recorded from all F0-males on the scheduled day of necropsy.Of the males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals in the control group and in the highest dose group.
- The additional slides of the testes of the males of the control group and the highest dose group, and all males suspected to be infertile, to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously.
- All gross lesions of all animals (all dose groups).
- Liver of the females of the control group and the highest dose group.
- The reproductive organs of males that failed to sire and females that failed to deliver healthy pups. Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

Postmortem examinations (offspring):

Pups surviving to the planned termination and the pup euthanized in extremis were killed by
decapitation between Days 5-7 of lactation.
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 (Ref. 4) 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.

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 on Day 4 of lactation / Number of pups born alive x 100.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 75 mg/kg, 2 males and 1 female died spontaneously within the first week of treatment (after resp. 4 days, 6 days and 2 days of treatment). Consequently, the dose level was lowered to 35 mg/kg from Day 7 onwards; no further unscheduled deaths occurred.
Flat posture was noted for 3 males and 4 females and piloerection was noted for 1 female during the first week of treatment when they were exposed to 75 mg/kg. These signs were seen for individual animals over limited occasions and there were no clinical signs noted for the animals that died spontaneously. When the dose level was lowered to 35 mg/kg, piloerection, lethargy, uncoordinated movements, hunched posture and salivation were noted for individual animals only on limited occasions. Piloerection was also noted for one animal at 5 mg/kg. At the limited incidence observed, these signs were not considered to be adverse. No other clinical signs were seen.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males and females in the highest dose group had lower body weights and lower weight gains or weight loss compared to controls on Day 7 of the treatment period when animals received 75 mg/kg. From Day 8 onwards, high dose animals received 35 mg/kg. Body weights and weight gains remained lower for males during the premating and mating periods after the dose level was lowered (absolute body weights were not significantly lower on Day 15 of the mating period), though they were partially recovering. Body weights and weight gains were also significantly lower for females at 35 mg/kg on Day 8 of the premating period and Day 1 of the mating period (gains were not significantly different during mating). However, female weights and weight gains normalized to that of control levels during the post coitum and lactation periods. Females at 35 mg/kg gained significantly more weight on Day 11 of the post coitum period. This was not considered to be toxicologically relevant as the effect was transient and lower weights/weight gains would be expected if this were reflective of treatment related toxicity.
Absolute and relative food consumption was lower for animals of both sexes during the first week of treatment at 75 mg/kg. Mean food consumption was approximately half that of control animals (range 48-58% of controls). After lowering the high dose to 35 mg/kg, food consumption before or after allowance for body weight recovered for high dose animals and remained similar to controls for the remaining duration of treatment, or even surpassed it (for males during the first week of the mating period). There were no other effects on food consumption for animals in other treatment groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The gestation index and duration of gestation were unaffected by treatment up to 35 mg/kg.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
On e control male was found with reduced size of the testes and epididymides. Microscopically, this male showed massive tubular atrophy of the testes and no sperm in the lumen of the epididymides; there were no normal spermatogenic stages present in the testes for this male and he did not sire a litter. As this was a control animal, it was in no way related to treatment with TMAHP. No other effects on sperm measures were observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. For one control female and one female treated at 5 mg/kg bw/ day, the number of pups delivered were slightly higher than the number of implantation sites. This was considered to be caused by normal resorption of these areas as these enumerations were performed on Day 5 of lactation. There were 9, 10, 10 and 9 pregnant females in the control, 5, 15 and 35 mg/kg groups, respectively. 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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Females at 35 mg/kg had higher absolute (25%) and relative liver weights (27%) than controls. Microscopically, more glycogen was found in 8 rats at 35 mg/kg/day, which was determined to be treatment related but not adverse. Relative kidney weights were higher for females at 15 mg/kg than controls (4%). In the absence of a dose response relationship, these were not considered to be toxicologically relevant. Terminal body weights and the weight of the testes, epididymides, liver and kidneys of treated males were similar to those of controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic findings noted for one male and one female at 75 mg/kg that were found dead included an enlarged liver, advanced autolysis and cannibalism. No macroscopic findings were seen for the second male found dead.
One female (highest dose group) had a total litter loss. Her macroscopic findings included pale discoloration of the liver and reduced size and tan discoloration of the thymus. One control animal was found with reduced size of the testes and epididymides. Microscopically, this male showed massive tubular atrophy of the testes and no sperm in the lumen of the epididymides; there were no normal spermatogenic stages present in the testes for this male and he
did not sire a litter. As this was a control animal, it was in no way related to treatment with TMAHP. All other findings seen for control and/or treated animals including nodule(s) on the epididymides, pelvic dilation of the kidneys, a tan focus, nodule or discoloration of the clitoral glands and enlarged
clitoral gland were incidental in nature.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There was a test item-related microscopic finding in the liver of females treated at 35 mg/kg/day: Increased glycogen was recorded in 2/10 females (minimal) of the Control Group and in 8/10 females treated at 35 mg/kg/day (4: minimal, 3: slight, 1: moderate). The higher liver weights were due to the variability inherent to their non-fasted and lactating status. The increased liver weights for high dose females were likely secondary to this and were not reflective of treatment related toxicity. There were no other test item-related microscopic findings in the organs examined. There was one pair of the control group and two pairs at 35 mg/kg/day that failed to sire or deliver healthy pups. All findings recorded in the reproductive organs of the remaining animals were within the normal range of background pathology. The spermatogenic staging profiles were normal for all males examined except for one control male.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight loss and mortality seen at 75 mg/kg bw/day.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
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. However, 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. 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 (OFFSPRING)
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 WEIGHT (OFFSPRING)
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 two smaller litters and was not considered treatment related or toxicologically relevant.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead or were killed in extremis included emaciated, advanced autolysis and no milk in the stomach. Incidental macroscopic findings among surviving pups included missing toe. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at 35 mg/kg bw/day.

Reproductive effects observed:

not specified

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.

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 reproductive 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 weight gains or weight loss compared to controls on Day 7 of the treatment period (coinciding with reduced food intake) and clinical signs including piloerection and flat posture, the highest dose was lowered to 35 mg/ jg bw/ day after the first week. No further mortality occurred. At 35 mg/kg bw/ day piloerection, lethargy, uncoordinated movements, hunched posture and salivation were noted for individual animals only on limited occasions. No other toxicologically relevant differences in body weight gain of treated groups compared to controls were noted. 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. No treatment-related changes were noted in the clinical appearance or macroscopic findings. No reproductive toxicity was observed up to the highest dose level tested (35 mg/kg). Based on these data, the parental and reproductive No Observed Adverse Effect Level (NOAEL) was found to be 35 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles (Klimisch 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was conducted according to OECD guideline 421 and according to GLP principles with tetramethylammonium hydrogen phtalate 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 weight gains or weight loss compared to controls on Day 7 of the treatment period (coinciding with reduced food intake) and clinical signs including piloerection and flat posture, the highest dose was lowered to 35 mg/ jg bw/ day after the first week. No further mortality occurred. At 35 mg/kg bw/ day piloerection, lethargy, uncoordinated movements, hunched posture and salivation were noted for individual animals only on limited occasions. No other toxicologically relevant differences in body weight gain of treated groups compared to controls were noted. 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. No treatment-related changes were noted in the clinical appearance or macroscopic findings. No reproductive toxicity was observed up to the highest dose level tested (35 mg/kg bw/day). Based on these data, the parental NOAEL was found to be 35 mg/kg bw/day and the reproductive NOAEL was found to be ≥35 mg/kg bw/day.

Short description of key information:
In a reproduction/developmental toxicity screening test of tetramethylammonium hydrogen phtalate in rats by oral gavage performed according to OECD guideline 421 and according to GLP principles, a parental NOAEL of 35 mg/kg bw/day and a reproductive NOAEL of ≥35 mg/kg bw/ day was established. The LOAEL parental was found to be 75 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
One study available.

Effects on developmental toxicity

Description of key information

In a reproduction/developmental toxicity screening test of tetramethylammonium hydrogen phtalate in rats by oral gavage performed according to OECD guideline 421 and according to GLP principles, a parental NOAEL of 35 mg/kg bw/day and a developmental NOAEL of ≥ 35 mg/kg bw/day was established. The LOAEL parental was found to be 75 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:

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

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

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.

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.

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.

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

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.

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles (Klimisch 1).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was conducted according to OECD guideline 421 and according to GLP principles with tetramethylammonium hydrogen phtalate. 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 bw/day 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 NOAEL was found to be 35 mg/kg bw/day and the developmental NOAEL was found to be ≥ 35 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
One study available

Justification for classification or non-classification

Based on the current data, tetramethylammonium hydrogen phtalate is not classified for effects on reproduction or development according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.