Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

publication

Title:

Assessment of the morphology and significance of the lymph nodal and hepatic lesions produced in rats by the feeding of certain mineral oils and waxes.

Author:

Carlton WW, Boitnott JK, Dungworth DL, et.al.

Year:

2001

Bibliographic source:

Exp Toxicol Pathol.53(4):247-55.

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA, OPPTS870.3650, Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in this report essentially conform to the following guidelines.

1. The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
2. OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
3. Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
4. OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
5. The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

EC Number:

615-240-7

Cas Number:

710292-85-6

Molecular formula:

Not available for this UVCB

IUPAC Name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Test material form:

other: waxy solid

Details on test material:

- Name of test material (as cited in study report): HK 128
- Substance description: red-violet waxy solid
- Storage condition of test material: at room temperature in the dark
- pH (1% in water; indicative range): 9.9-10.0
- Stability at higher temperatures: yes, max. temperature 100 °C for a maximum of 2 hours

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: within ± 20% of the sex mean (males:280-309 grams, females: 191-219 grams)
- Fasting period before study: no
- Housing: groups of 5 animals/sex/cage during pre-mating and post-mating (males), females were individually housed post-mating and during lactation; Macrolon plastic cages with sterilized sawdust as bedding material and paper as cage-enrichment/nesting material
- 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): 18.7-20.9
- Humidity (%): 38-95; temporary deviations from the minimum and daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 01 May - 22 June 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

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. No correction was made for the purity of the test substance. Adjustment was made for specific gravity (1.036) of the vehicle. The test substance was warmed to facilitate mixing (max temperature= 82.7°C, for a maximum of 58 minutes).
Storage conditions of formulations: at ambient temperature
Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight
Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis were conducted twice during the main study (Days 2 and 14 of the treatment phase; 02 and 14 May 2012 respectively), according to a validated method (Project 498662). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

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 85% and 115%).
A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use on Day 14. The maximum contribution based on area to the other samples was 3.3%. A small response was also seen in the blank injection sample analyzed. In the formulations of Group 1 of Day 2, no test substance was detected.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours (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.

Duration of treatment / exposure:

Females were exposed for 42-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female from Group 1, 2 and 4 each were not dosed during littering.

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. Animals were dosed up to the day prior to scheduled necropsy.

Duration of test:

42-52 days

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, locomotor activity, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. 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). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals. Observations were started after dosing at no specific time point, but within a similar time period for the respective animals throughout the study. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on
Days 1 and 4.

FOOD CONSUMPTION: Yes.
Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller,
Austria) prepared with EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00
and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/group
- Parameters checked were: according to OECD 422 (1996)

CLINICAL CHEMISTRY: Yes
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller,
Austria) prepared with Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood
sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00
and 10.30 a.m
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/group
- Parameters checked in table were: according to OECD 422 (1996); except alkaline phosphatase was measured instead of sorbitol dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity
test (recording period: 1-hour for individual animals, using a computerised monitoring system. During the motor activity
test, males were caged individually and females were caged with their pups.

GROSS PATHOLOGY: Yes
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was
provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using
isoflurane vapor and subsequently exsanguinated.
Necropsy was conducted on the following days:
Females which delivered: lactation Days 5-67
Females which failed to deliver: post-coitum Days 26-27 (females with evidence of mating)
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs,
with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were
recorded. The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/group and all animals that were killed in extremis : according to OECD 422 (1996)
- All remaining animals and females which failed to deliver: according to OECD 422 (1996)

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled
day of necropsy:
- Selected 5 animals/group: according to OECD 422 (1996)

HISTOPATHOLOGY: Yes, according to OECD 422 (1996).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
- Clinical signs: At least once daily, detailed clinical observations were made in 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.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated. The stomach was examined for the presence of milk.

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.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the 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. 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 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

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY:
One female at 1000 mg/kg was euthanized in extremis on Day 8 of the treatment period. She was noted with lethargy, hunched posture, labored respiration, piloerection, lean appearance, ptosis, chromodacryorrhoea, and hypothermia; she also had severe body weight loss (of 22%, on Day 8 of the pre-mating period) prior to euthanasia. This female was found with her stomach distended with gas, with a dark red focus on the stomach glandular mucosa, both adrenal glands enlarged, reduced size of the thymus and was found to be emaciated at the macroscopic examination. At the microscopic examination, necrosis of the caecum villi and of liver hepatocytes were seen, along with lymphoid atrophy. A relationship to treatment could not be excluded.

CLINICAL SIGNS:
There were no clinical signs of toxicity noted during the observation period for surviving animals.
Salivation was noted for all surviving females at 1000 mg/kg through most of the treatment period, beginning around Day 10 of treatment (or slightly later). Salivation was also noted for 6 of the 10 females at 300 mg/kg. Due to its time of occurrence (after dosing), salivation was likely due to a physiological response attributable to the taste or possible irritancy of the test substance, was not considered to be a sign of toxicity.

NEUROBEHAVIOUR:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHT:
No toxicologically relevant changes in body weights and body weight gain were noted.
Females at 1000 mg/kg had significantly higher body weight gain on Day 1 of the mating period. However, the difference from controls was only slight, and was not considered to be attributable to treatment with HK 128.

FOOD CONSUMPTION:
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted up to 1000 mg/kg.
Relative food consumption was slightly higher for males and females at 1000 mg/kg over Days 1-8 of the mating period and Days 8-15 of the pre-mating period, respectively. This was not considered toxicologically relevant as the difference from controls was slight, values remained within the range considered normal for animals of this age and strain, and a reduction in food consumption would be more likely to be seen if the difference from controls was due to toxicity.

HAEMATOLOGY:
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The significant increase in reticulocytes seen for females at 1000 mg/kg was attributable to a high value obtained for one female, and was not indicative of a treatment-related effect.
The mean neutrophils were higher and mean lymphocytes lower for controls than for all treated females. However, high lymphocytes with concomitant low neutrophils were also noted for a control female, which contributed to these mean values and high standard deviation seen for control females in these two parameters. When re-calculated without her data, control means for neutrophils and lymphocytes were the same for control females as for the treated groups.

CLINICAL BIOCHEMISTRY:
There were no toxicologically relevant effects on clinical biochemistry parameters up to 1000 mg/kg.
Females at 1000 mg/kg had significantly higher cholesterol values compared to controls. In the absence of effects on other endpoints indicating altered liver function, these were not considered to be biologically significant.
The significantly lower total protein and albumin seen for females at 100 mg/kg were likely due to slightly low values obtained for two female. These changes occurred in the absence of a treatment-related distribution and were not considered to be toxicologically relevant.

MACROSCOPIC EXAMINATION:
No treatment-related effects were noted at macroscopy.
Incidental findings seen for control and/or treated animals included pelvic dilation of the kidneys, reddish or dark red foci on the thymus or stomach glandular mucosa, reddish, red-brown, dark red, or tan discoloration of the thymus, clitoral glands and/or mandibular lymph nodes, yellowish, soft nodule on the head of the right epididymis, and scabbing on the cheek. These findings occurred for control and treated animals without a treatment related distribution and at the incidence observed, were not considered to be treatment related.

ORGAN WEIGHTS:
At 1000 mg/kg females had higher absolute and relative liver weights while higher relative liver weights were noted for males. These differences in organ weights and organ to body weight ratios were not accompanied by any treatment-related changes noted at the histopathological examination. As such these effects were considered treatment related but not toxicologically relevant.

MICROSCOPIG EXAMINATION:
Microscopic treatment-related findings were present in females in the mesenteric lymph node including necrosis that was present in 2/5 females (1 minimal, 1 slight) treated at 1000 mg/kg (Group 4). Additionally, macrophage foci were present at an increased incidence and severity in 5/5 female (2 slight, 3 moderate) rats treated at 300 mg/kg (Group 3) and in 5/5 female (1 slight 4 moderate) rats treated at 1000 mg/kg compared to 1/5 female (minimal) controls (Group 1) and 3/5 female (3 minimal) 100 mg/kg (Group 2) treated rats. A minimal degree of macrophage foci is considered to be within the normal limits.

REPRODUCTIVE DATA:
No toxicologically relevant effects on reproductive parameters were noted. There were 9, 10, 9 and 8 pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no treatment related effects on the mating, fertility and conception indices, precoital time, number of corpora lutea or implantation sites. The number of implantation sites at 300 mg/kg were significantly lower than for females of the control group. In the absence of a treatment related distribution and any relevant effects on other reproductive parameters, this was not considered to be treatment-related.

GESTATION:
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg bw/d.

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. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS
One pup of the control group and two, one and one pups of the 100, 300 and 1000 mg/kg groups were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups 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 PUPS
Pale appearance was noted for one pup at first litter check from one litter at 100 mg/kg. This was the only observation noted for any pup. The nature and incidence of this remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

BODY WEIGHT PUPS
Body weights were significantly higher for pups of both sexes at 300 mg/kg on lactation Days 1 and 4. The increased body weights were, in part, attributable to very high weights obtained from two litters with 8 and 6 pups, respectively. The relatively low number of pups in these litters contributed to the higher weights as more milk was available for each individual pup than in larger litters.

MACROSCOPY PUPS
No effects observed.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to the highest dose tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for parental toxicity is set at 100 mg/kg bw/d based on an increased incidence and severity of macrophage foci at 300 and 1000 mg/kg bw/d with concurrent increased incidence and severity of necrosis of the mesenteric lymph node at 1000 mg/kg bw/d. The NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/d in the absence of effects observed.

Executive summary:

In an OECD 422 study rats were administered 0, 100, 300 or 1000 mg/kg bw/d of HK 128 by gavage. Parameters checked were according to OECD 422. No effects were observed with relation to body weight, food consumption, neurobehavioural examination, haematology and macroscopy. One female at 1000 mg/kg bw/d was killed in extremis on day 8 of treatment having severe body weight loss, stomach distended with gas, with a dark red focus on the stomach glandular mucosa, both adrenal glands enlarged, reduced size of the thymus, necrosis of the caecum villi and of liver hepatocytes along with lymphoid atrophy. At 1000 mg/kg bw/d females had significantly higher cholesterol values compared to controls. In the absence of effects on other endpoints indicating altered liver function, these were not considered to be biologically significant. At 1000 mg/kg bw/d absolute and relative liver weight was increased in females. In absence of histopathological effects on these organs these effects were not considered to be toxicologically relevant. At 300 and 1000 mg/kg bw/d an increased incidence and severity of macrophage foci in the mesenteric lymph node was noted with concurrent increased incidence and severity of necrosis of the mesenteric lymph node at 1000 mg/kg bw/d. Based on the effects on the mesenteric lymph node, the NOAEL is set at 100 mg/kg bw/d for parental toxicity. As no effects on developmental toxicity were observed the NOAEL was established to be 1000 mg/kg bw/d.