Diisopropylbiphenyl and triisopropylbiphenyl

Endpoint summary

Description of key information

Repeated dose toxicity study in the rat (oral, 28 days, OECD 407): LOAEL 35 mg/kg bw/day

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, extended and comprehensive study regime

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

test group sizes, histology and clinical biochemistry extended status (TSH, T3, T4)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. /NL [RccHanTM: WIST(SPF)]
- Age at study initiation: 7 weeks
- Weight at study initiation: 249-253 g (m); 168-173 g (f) (Report, Section 8: Summary Tables)
- Fasting period before study: no
- Housing: Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Solution in corn oil

VEHICLE: Corn oil
- Justification for use and choice of vehicle (if other than water): Corn oil is non-toxic and miscible with the test substance.
- Concentration in vehicle: 7, 30 and 120 g/L
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of Dose Formulations:
One week at room temperature (20 ± 5 °C) based upon the results of stability analyses performed within the Harlan Laboratories study D66425 (non-GLP) and confirmed within this study.

Storage of Dose Formulations:
At room temperature (20 ± 5 °C) in glass beakers, away from direct sunlight and protected from UV light.

The samples were stored refrigerated (ca. 5 ± 3 °C) and light protected until delivery to the analytical laboratory or delivered directly at ambient temperature (20 ± 5 °C) and light protected and stored there refrigerated (ca. 5 ± 3 °C) and light protected until analysis.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 35, 150, and 600 mg/kg bw/d
Basis:
actual ingested

Remarks:

Doses / Concentrations:
35, 150, and 600 mg/5 mL
Basis:
other: concentration in the vehicle

No. of animals per sex per dose:

10 in control and test groups / 5 in recovery groups (control and treated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on a previous 7-day dose range finding study (Harlan Study Number D66425, non-GLP) with the same test item.
There were significant treatment-related changes in organ weights of rats dosed >= 300 mg/kg bw/day as well as distinct decreases in food
consumption of rats administered 1000 mg/kg bw/d.
- Rationale for selecting satellite groups: post-exposure recovery of treatment-related effects
- Post-exposure recovery period in satellite groups: 14 d

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1x/d (acclimatisation period); 2x/d on days 1 to 3; 1x/d thereafter (treatment period) / 1x/d (recovery)
- Cage side observations checked in Chapter 9, p. 184 - 188) (Treatment), were included.

DETAILED CLINICAL OBSERVATIONS: Yes (see Report 3.7.1)
- Time schedule: 1x/wk (acclimatisation period) / 1x/wk (during weeks 1-3, in week 4 included in FOB = Functional Observational Battery)/
none (recovery)
- observations checked in Chapter 9, p. 185 - 188) (Treatment), were included

BODY WEIGHT: Yes (see p. 104 and 243, Figures p. 61)
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: after week 4 (control and treated groups), after week 6 (recovery control and high-dose group, recovery)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 13-14 at wk 4, 4 at wk 6 (Control); 8-9 at wk 4, 4 at wk 6 (treated groups)
- Parameters in Tables p. 117 and 274.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after week 4 (control and treated groups), after week 6 (recovery control and high-dose group, recovery)
- Animals fasted: Yes
- How many animals: 15 at wk 4, 4 or 5 at wk 6 (Control); 10 at wk 4, 4 or 5 at wk 6 (treated groups)
- Parameters in Tables p. 129 and 324

Determination of thyroid hormones (T3 = trijodinethyronine, T4 = thyroxine/tetrajodinethyronine) and the pituitary hormone TSH (= thyroid-stimulating hormone/thyrotropin) in blood: all control and dose groups (see Report 3.10 and Appendix VI, p. 436): The samples were stored frozen
at approximately -80 °C and protected from light until analyses were performed.

URINALYSIS: Yes
- Time schedule for collection of urine: after week 4 (control and treated groups), after week 6 (recovery control and high-dose group, recovery)
- Metabolism cages used for collection of urine: Yes
- Parameters in Tables p. 137 and 326

NEUROBEHAVIOURAL EXAMINATION: Yes (see Report 3.7.2)
- Time schedule for examinations:
- Dose groups that were examined: all
- Battery of functions tested (modified Irwin screen test): grip strength / motor activity in week 4

OTHER:
- A vaginal smear was taken daily during the last two weeks of treatment from all females, and the stage of estrus was evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
At NECROPSY, (4 wks: end of treatment; 6 wks: end of recovery), a blood sample was taken from each animal by heart puncture (ca. 2 mL).
Following centrifugation between 2 °C and 8 °C, the serum was divided in 2 aliquots of at least 350 µL and transferred to plastic (polypropylene) tubes.
These samples were stored at approximately -80 °C and protected from light prior to transfer for analysis of hormone activity.

HISTOPATHOLOGY: Yes (see examined tissues in Table below "Other information...").
Furthermore, from the animals of the low dose groups, liver, lung, stomach, thyroid, pituitary gland, prostate gland, coagulating glands, seminal vesicles, ovaries, uterus and vagina were additionally examined due to findings in the high dose group (see special histopathology report Appendix VII).

Other examinations:

Stages of spermatogenesis and histopathology of interstitial cell structure in all males of group 1 and 4 at the end of treatment.

Statistics:

·The Dunnett-test (many to one t-test) based on a pooled variance estimate;
·The Steel-test (many-one rank test) instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
·Fisher's exact-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In males and females of groups 3 and 4 salivation was noted towards the end of the treatment period. Otherwise, no test item-related clinical signs were noted during the treatment phase.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were statistically significantly decreased in males of group 4 compared to control group on day 8 and remained slightly lower until the end of the treatment phase. Body weight gain (in %) in males of group 4 was markedly reduced compared to males from the control group during the whole treatment phase. Mean body weights of test item-treated females were comparable to control values, whereas body weight gain was increased compared to control values attaining statistical significance on days 15 and 22.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative food consumption was statistically significantly lower compared to control group in group 4 males and females on days 1 - 8. Thereafter, food consumption was increased compared to control values in males and females of this group, occasionally attaining statistical significance, until the end of treatment. On days 22 - 28, absolute food consumption was statistically significantly increased compared to control also in females of groups 2 and 3 in a dose dependent manner, relative food consumption was statistically significantly increased also in females of group 3.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in white blood cell parameters were noted in males of group 4 and in test item-treated females of all dose levels including increased total white blood cell count (only females) and increased numbers of monocytes and lymphocytes. Furthermore, the prothrombin time (PT, rel. 1) was increased in all test item-treated males and in females of group 4. At thje end of the recovery period, PT was significantly increased in males of group 4.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol, phospholipids, triglycerides increased / proteins, albumin increased / globulin decreased

After 4 weeks of treatment, the TSH-levels were statistically significantly increased in males and females of group 3 and 4 compared to control animals. T3 levels were statistically significantly decreased in males and females of group 3 and 4 compared to the control group. The T4 levels were statistically significantly decreased in males of group 3 and 4 and non-significantly decreased in females of group 4.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Mean locomotor activity was statistically significantly decreased in males of group 4 from the 10 to 20-minute interval to the end of the measurement period after 60 minutes and in males of group 3 from the 40 to 50-minute interval to the end of the measurement. In group 4, also the total mean locomotor activity was statistically significantly lower compared to control.

In the females, a similar trend was noted in group 3 and 4 from 40 to 60 minutes but the total mean locomotor activity was not affected.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver and thyroid weights were increased in males and females of all dose groups in a dose-dependent manner at the end of the treatment phase. The organ weights of prostate gland / seminal vesicles and epididymides were statistically significantly decreased in males of group 4. In females, weights of ovaries were decreased in group 4 and weights of uterus were decreased in groups 3 and 4. A slight increase in kidney weight, not clearly dose-related, was noted in the males of all dose groups and in females of group 4. Increased adrenal weights were noted in males of group 4.

At the end of the recovery period, liver weights were still slightly increased in males and females of group 4 compared to control animals. The thyroid weight in high-dose females normalized to control level, while in males the weight increment did not fully regress during the 14 days of recovery. Effects on prostate gland / seminal vesicles and ovaries were not fully reversible within the post-exposure period. Weight increases of the adrenal gland seen in high-dose males were reversible. However, a slight decrease in the adrenal weight was noted in the females of group 4 after recovery.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged livers were noted at necropsy in one male of group 2, in all males of group 3 and in all males and females of group 4. Furthermore, in all males of group 4, prostate gland and seminal vesicles were reduced in size and in one male also epididymides were reduced in size macroscopically.
After 2 weeks of recovery, the liver of one male was still macroscopically enlarged. These findings were in correlation with organ weight changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related histopathological findings were observed:

- Hepatocyte hypertrophy (males of all dose groups and females of groups 2 and 4 ) and necrosis (males of all dose groups, females group 4) at minimal to moderate grade, still present primarily in males at minimal degree after recovery.
- Follicular cell hypertrophy/hyperplasia in the thyroid at minimal to moderate grade (males and females of all dose groups), decreasing but still present in all males and one of five females after recovery.
- Hypertrophic/vacuolated cells in Pars Anterior of the pituitary gland at a minimal and slight degree of severity (males and females group 4), decreasing but still present in males after recovery.
- Testicular Leydig cell atrophy of minimal grade, prostate, seminal vesicle and coagulating gland atrophy of slight to moderate degree (males group 4), fully reversible after the recovery period.
- Follicular degeneration in ovaries of minimal grade and disturbance of estrus cycle (females group 4), fully reversible after the recovery period.
- Low-grade erosion and cystic/dilated glands in the glandular stomach (males and females group 4), fully reversible after the recovery period.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No differences in oestrus stage were noted between the groups through examination of vaginal smears.

Key result

Dose descriptor:

LOAEL

Effect level:

35 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

ovary

testes

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

ORGAN WEIGHTS

The following differences in organ weights between test item-treated animals and control animals after 4 weeks of treatment and additional 2 weeks of recovery were considered to be test item-related as they were statistically significant and either dose-related and/or consistent over time and/or between sexes:

Table 1 (Report Table 6)  Changes in Mean Absolute and Relative Organ Weights Considered to be Test Item-related (in Terms of Percentages Compared to Controls)

Dose Level mg/kg bw/day	Males			Females
	35	150	600	35	150	600
Liver weight	+39.8%**	+88.0%**	+137.5%**	+26.1%**	+71.7%**	+165.3%**
Liver / bw ratio	+32.2%**	+79.9%**	+156.0%**	+21.5%**	+72.2%**	+170.3%**
Thyroid weight	+26.3%°	+52.6%**	+89.5%**	+11.8%°	+41.2%**	+70.6%**
Thyroid / bw ratio	+16.7%°	+50.0%**	+100.0%**		+33.3%**	+66.7%**
Thymus weight						-18.6%°
Thymus / bw ratio						-17.3%°
Kidney weight	+11.1%*	+14.1**	+5.0%°			+7.0%°
Kidney / bw ratio	+4.9%°	+8.2%*	+13.1%**			+9.2%*
Adrenal weight			+34.4%**
Adrenal /bw ratio			+42.1%**
Prost/Semi weight		-10.0%°	-52.3%**
Prost/Semi / bw ratio		-14.5%°	-49.0%**
Epididymides weight			-12.7%**
Epid. / bw ratio		-6.0%°	-6.3%°
Ovaries weight						-23.0%**
Ovaries /bw ratio						-22.0%**
Uterus weight					-23.1%*	-23.1%*
Uterus / bw ratio					-23.9%*	-21.7%°
Recovery
Liver weight			+14.3%°			+14.1%*
Liver / bw ratio			+18.2%**			+19.3%*
Thyroid weight			+42.9%*
Thyroid / bw ratio			+50.0%*
Adrenal weight						-10.5%*
Adrenal / bw ratio
Prost/Semi weight			-21.6%*
Prost/Semi / bw ratio			-19.2%°
Ovaries weight						-10.8%°
Ovaries /bw ratio
* statistically significant p<0.05; ** statistically significant p<0.01; ° no statistical significance -bw = body weight; Epid. = Epididymides; Prost/Semi = prostate gland / seminal vesicles -blank cells = no relevant changes noted compared to control.

 

HISTOPATHOLOGY / MICROSCOPIC FINDINGS

Test item-related microscopic findingswere noted in the liver, thyroids, pituitary, testes, prostate, coagulating glands, seminal vesicles, ovaries, uterus, vagina and stomach. The observations in liver and thyroid gland are summaries in Table 2 and 3 below. See Attached Document for the summary of all findings.

Severity codes for evaluating histopathological findings are as follows:

GRADE 1 = Minimal / very few / very small

GRADE 2 = Slight / few / small

GRADE 3 = Moderate / moderate number / moderate size.

  Table 2 (Report p. 42/43): Microscopic changes recorded in LIVER at the end of the treatment and recovery period

Sex	Male				Female
Dose-level (mg/kg bw/day)	0	35	150	600	0	35	150	600
after 4 weeks (end of treatment)
Number examined	10	10	10	10	10	10	0	10
- Hypertrophy; diffuse	0	1 (1.0)	9 (1.8)	9 (2.1)	0	2 (1.0)	-	10 (2.4)
- Hypertrophy; centrilobular	0	9 (1.1)	1 (2.0)	1 (2.0)	0	7 (1.0)	-	0
- Necrosis, single cell	0	3 (1.0)	3 (1.0)	6 (1.3)	0	0	-	3 (1.3)
- Necrosis, focal	0	0	0	0	0	0	-	2 (3.0)
after 6 weeks (with recovery of 2 weeks)
Number examined	5	0	0	4*	5	0	0	5
- Hypertrophy; centrilobular	0	-	-	3 (1.0)	0	-	-	1 (1.0)
- Necrosis, single cell	0	-	-	2 (1.0)	0	-	-	0

( ) mean severity    /   Findings in bold are considered treatment-related.

*       High dose male 50 died before the recovery period, and has therefore been excluded from the incidence table.

----------------------------

Hepatocyte hypertrophy was characterized by diffuse or centrilobular enlargement of hepatocytes, with granular eosinophilic cytoplasm in centrilobular hepatocytes at higher severity grades. Livers of males receiving 150 mg/kg/day were examined because of macroscopic findings. Livers of females receiving 150 mg/kg/day were not examined.

Table 3 (Report p. 43): Microscopic changes recorded in the THYROID at the end of the treament and recovery period

Sex	Male				Female
Dose-Level (mg/kg bw/day)	0	35	150	600	0	35	150	600
after 4 weeks (end of treatment)
Number examined	10	10	10	10	10	10	10	10
- Hypertrophy/ hyperplasia; follicular cell	1 (1.0)	5 (1.2)	8 (1.5)	10 (2.0)	0	5 (1.0)	6 (1.0)	10 (2.0)
after 6 weeks (with 2 weeks of recovery)
Number examined	5	0	0	4^*	5	0	0	5
- Hypertrophy/ hyperplasia;  follicular cell	0	-	-	4 (1.0)	0	-	-	1 (1.0)

( ) mean severity   /  Findings in bold are considered treatment-related.

* High dose male 50 died before the recovery period, and has therefore been excluded from the incidence table.

THYROID HORMONES

Table 4 (Report Tab. 5): TSH, T4 and T3 Levels in Blood Serum of Male and Female Rats at Termination of

Exposure to DIPB (28 Days) and Recovery (14 Days)

Group [DIPB in mg/kg bw/d]	TSH[µIU/mL]		T4[µg/dL]		T3[µg/dL]
	Exposure	Recovery	Exposure	Recovery	Exposure	Recovery
	Mean ± SD		Mean ± SD		Mean± SD
Males
1    [0]	0.29± 0.36	0.58± 0.53	4.4± 0.7	4.3± 0.6	0.075± 0.015	0.073± 0.010
2  [35]	0.51± 0.58	---	4.2± 0.8	---	0.070± 0.012	---
3 [150]	3.25± 3.33*	---	2.9± 0.8**	---	0.056± 0.011**	---
4 [600]	2.91± 2.35*	0.62± 0.23	2.5± 0.6**	5.0± 0.7	0.051± 0.007**	0.081± 0.005
Females
1    [0]	0.28± 0.25	0.36± 0.19	2.7± 0.8	2.3± 0.8	0.079± 0.008	0.084± 0.013
2  [35]	0.51± 0.32	---	3.5± 1.0	---	0.067± 0.016	---
3 [150]	4.68± 5.49*	---	3.5± 0.8	---	0.059± 0.018**	---
4 [600]	7.57± 5.74**	0.53± 0.35	2.0± 0.3	3.2± 0.8	0.046± 0.007**	0.089± 0.016
*      Significantly different from control (group 1): Dunnett´s test, significance level p <0.05 **    Significantly different from control (group 1): Dunnett´s test, significance level p <0.01 Note: Values are rounded up to the previous decimal place (see detailed tables in Report, Appendix VI on p. 437). High standard deviations (SD), indicators of high variance, are often attributable to only one or two relatively high values in the series of 10 samples (see detailed tables in Report, Appendix VI on p. 437).

Conclusions:

Based on the results of this study and the occurrence of adverse effects on the liver (hepatocellular hypertrophy and necrosis) and thyroid gland (hypertrophy/hyperplasia of follicular cells) also in the low dose group (35 mg/kg bw/day), a no observed adverse effect level (NOAEL) of the test item could not be established after 4 weeks of oral treatment at dose levels of 35, 150 and 600 mg/kg bw/day.

There was no evidence of histopathological effects on the male and female reproductive system except for the high dose level (low-grade atrophy or degeneration), which proved to be reversible within the post-exposure period of 14 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

35 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6.1, of Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

liver

thyroid gland

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

Repeated dose toxicity: oral

A 28 day study in rats receiving Alkylation and transalkylation products of biphenyl with propene (EC No. 915-589-8) by gavage in corn oil was conducted (Simon, 2014). The test item proved to be a potent inducer of liver growth, which - to a high extent - is considered to be an adaptive response to the metabolic burden, also associated with shifts in various haematological and blood-chemical parameters. Slight degenerative changes are evidenced by occasional dose-related hepatocellular necrosis ("single-cell necrosis"). Effects were observed on testes and ovaries at the high dose, but oestrus cycles were not affected. Other relevant organ effects relate to the thyroidal hypertrophy/hyperplasia associated with disturbance of thyroid-hormone balance. The most prominent results of this investigation in rats are consistent with the hypothesis that the induction of hypertrophy and hyperplasia in the thyroid is an indirect disrupting rather than a direct stimulatory/inhibitory effect of the test item within the hypothalamo-pituitary-thyroid axis: Secondary to the microsomal enzyme-induced increase in the hepatic biotransformation and excretion of the thyroid hormones, basically T4, the simple feedback turn-down mechanism on TRH release from the hypothalamus and TSH secretion from the pituitary are interrupted, while the TSH level continues increasing and stimulating the thyroid to compensate for losses in T3 and T4. The symptoms are characteristic of hypothyroidism/thyroid hypofunction. The toxicological significance for humans has to be critically considered, since empirical evidence suggests that the presumptive mode of action underlying in rodents is unlikely to be operative to that expressive extent in primates.

As histopathological effects were noted in the liver and thyroid at all dose levels, a NOAEL could not be determined in this study. The LOAEL is considered to be 35 mg/kg bw/day.

Justification for classification or non-classification

Hepatic and thyroid effects were noted at all dose levels including the low dose leverl of 35 mg/kg bw/day. Accordingly a NOAEL could not be established in this study.

Based on these results, the test substance is considered to meet the classification criteria for a specific target organ toxicity (STOT RE 2) according to Regulation (EC) No. 1272/2008 (CLP).