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

Endpoint summary

Description of key information

A 28-day repeated dose toxicity study in rats was undertaken with HK 128 at doses of 100, 300 and 1000 mg/kg bw.  The NOAEL for adult 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.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650, Combined repeated dose toxicity study with the reproduciton/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

Species:

rat

Strain:

Wistar

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. 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

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral 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%).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. 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. Female nos. 49 (Group 1), 54 (Group 2) and 75 (Group 4) 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.

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 10-day dose range finding study (Project 498660), in which females (3/dose) were administered 500 or 1000 mg/kg bw/d by gavage. No mortality, no clinical signs, no effect on food consumption, macroscopic examination and liver and kidney weight was observed. A slight body weight loss for 2 females on day 5 that recovered by day 10 was noted at 500 mg/kg bw/d only.
Based on the results of the range-finding study, dose levels of 100, 300 and 1000 mg/kg bw/d were used in the main study. Since no clinical signs were observed at a dose of 500 and 1000 mg/kg bw/d, clinical observations in the main study were conducted immediately after dosing.
- 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.

Positive control:

not applicable

Observations and examinations performed and frequency:

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: 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.

FOOD CONSUMPTION: Yes.
Weekly, for males and females. Food consumption was not recorded during the mating period. 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/sex/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/sex/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 males were tested during Week 4 of treatment and the selected
females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/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 (nos. 46, 66 and 77): post-coitum Days 26-27 (females with evidence of mating)
Males: following completion of the mating period (a minimum of 28 days of dose administration).
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/sex/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/sex/group: according to OECD 422 (1996)
- All remaining males: testes and epididymides

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

Sacrifice and pathology:

see above

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.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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 males and 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 all males and 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.
There was a trend towards higher neutrophils and lower lymphocytes (not statistically significant) for males at 1000 mg/kg. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and was not toxicologically relevant. 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 (no. 49), 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.
At 1000 mg/kg significantly higher alanine aminotransferase (ALAT) and inorganic phosphate were seen for males along with significantly lower total protein and albumin. Females at this dose level 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 females. Along with the lower potassium seen for males at 100 mg/kg, 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. Additionally, males at this dose level had higher relative adrenal weights and lower absolute and relative seminal vesicle weights. 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 males and females in the mesenteric lymph node including necrosis that was present in 3/5 males (1 minimal, 2 slight) and 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 male (3 slight, 2 moderate) and 5/5 female (2 slight, 3 moderate) rats treated at 300 mg/kg (Group 3) and in 5/5 male (3 slight, 2 moderate) and 5/5 female (1 slight 4 moderate) rats treated at 1000 mg/kg compared to 2/5 male (2 minimal) and 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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: increased incidence and severity of necrosis of the mesenteric lymph node was noted for animals at 1000 mg/kg while an increased incidence and severity of macrophagic foci in the mesenteric lymph node was noted for animals at 300 and 1000 mg/kg

Organ:

lymph node

Treatment related:

yes

Dose response relationship:

yes

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.

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 significantly higher alanine aminotransferase (ALAT) and inorganic phosphate were seen for males along with significantly lower total protein and albumin. Females at this dose level 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, while relative liver weights were higher in males. Males at this dose level had also lower absolute and relative seminal vesicle weights and a higher relative adrenal weight. 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Adequate

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Lymphadenopathy with enlarged nodes and congestion has been associated with exposure to industrial chemicals and medications. Infiltration of the abdominal lymph nodes by metabolite-laden macrophages is observed in lipid storage disorders in humans (Gaucher disease or Niemann-Pick disease, Medical Disability Guidelines). Administration of various mineral oils and waxes to Fischer 344 and Sprague-Dawley rats is known to result in lesions, primarily microgranulomas/granulomas, in the mesenteric lymph nodes and liver, with infiltratration of mononuclear inflammatory cells at various sites (Carlton, et.al, 2001). Thus, the finding in this study of macrophagic inflammatory cells in the mesenteric lymph node after repeated dosing of a lipophilic test material is best described as a physiological attempt to metabolize or remove material from the hepatic/systemic circulation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
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.

Repeated dose toxicity: via oral route - systemic effects (target organ) other: all gross lesions and masses

Justification for classification or non-classification

There is insufficient justification for classification for repeated dose toxicity, due to the findings of the 28-day repeated dose toxicity study of HK 128. Findings are typical in rats after repeated oral dosing of a lipophilic wax.