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Description of key information

Oral 90 d study (key), rats (10/sex/dose), Results: decreased feed consumption + body weights + testes weight, increased lethality +  weights of livers/kidneys, occurrence of hematuria + kyphosis + hydronephrosis + renal tubular and papillary necrosis + obstructive urinary tract lesions. A NOAEL could not be determined. LOAEL = 100 ppm (6 mg/kg bw/d for m, 8 mg/kg bw/d for f). 
Dermal 90 d study, rat, diethanolamin (DEA) salt of 4-tert-butylbenzoic acid tested. Results: Microcytic hypochromic anemia (animals of the two top doses); erythrocytic microcytosis at normal erythrocyte counts (at two low conc.), general dose-related increase in relative and absolute hepatic and renal weights. Decreased testis weight, sperm counts + LDH-X enzyme activity (males, two highest conc.). Testicular effects in males exposed daily to ≥70 mg/kg. LOAEL = 17.5 mg/kg bw/day
Dermal 28 d study, rat. Results: reduced growth rates/body weights in females (30 and 60 mg/kg bw/d), increased liver weights, increased weights of kidneys (two top doses, females), decreased testes weights (60 mg/kg/d, males). LOAEL = 7.5 mg/kg bw/d. (LOAEL = 7.5 mg/kd bw/d corresponds to 2.3 mg/kg bw/d in a 90d study - EU risk assessment (2009), p.64)
Inhalation 5 d study, rat. Results: no PTBBA-related effects found.
Inhalation 28 d study, rat. Results: increased liver weights (highest dose, f), increased body tremors + decreased activity + rearing activity + more facial staining, hair loss (high dose, m), urinating/defecating frequency affected (m), no lesions (organs/nervous tissue). NOAEC = 5 mg/m3
Inhalation 10/11 d study, rat. Results: unscheduled deaths, abnormal neurobehaviour, decrease of weight of body, testes and sperm count, increase of weights of liver and kidneys, lesions of kidneys, livers, spinal cord, central nervous system, testes, epididymides and thymus. No NOAEC was delivered; LOAEC = 12.5 mg/m3 (6 h/d, 7 d exp.) = 1.5 mg/m3 (extrapol. to 90d, see discussion)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: data is contained in EU risk assessment report of the substance - credibility is assumed. Reference to origin (Hunter et al.)
Reference:
Composition 0
Composition 0
Principles of method if other than guideline:
90 d repeated dose oral toxicity study with rats (male/female), uptake of the test substance with the diet, examination of substance-induced effects.
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rat
Strain:
other: Carworth farm
Sex:
male/female
Critical effects observed:
not specified

Unscheduled deaths of 9 of ten high dose males occurred by day 34, all females receiving 10000 ppm died by day 53. From the group receiving 3160 ppm, two males died by day 42 and six further males were killed moribund. Two mortalities and one female rat to be killed were also seen in the female group at 3160 ppm. Hematuria has been observed in one male and two females receiving 3160 ppm. Hind limb paralysis was reported for one male and one female exposed to diet concentration of 3160 ppm and one female at 1000 ppm. Kyphosis was observed in three rats receiving 3160 ppm and suspected to occur secondarily to chronic renal failure.

Final body weights were significantly depressed in males at diet concentrations of 316 and above and in female rats at 1000 ppm and above. The feed consumption was reduced in the two top doses to 50-70% of the control values and was not affected in the other dose groups. No treatment-related effect was seen on hematology parameters other than reduced erythrocyte counts in the surviving male treated with 10000 ppm and a shift towards increased percentages of neutrophils and reduction in lymphocyte counts at diet concentrations of 3160 ppm (surviving males and females) and of 10000 ppm (1 male survivor). Clinical chemistry findings showed reduced levels of total protein for male groups receiving 100 to 1000 ppm; urea concentrations were increased in males and female rats at diet concentrations ≥1000 ppm in a dose-related fashion.

Urinalysis revealed increased urine volume and reduced urine osmolality in rats treated with diet concentrations at 3160 ppm and above; protein concentrations were elevated in animals the 10000 ppm dose groups.

Relative organ weights of the liver and the kidneys increased at all diet concentrations. The testes to body ratio decreased in all male dose groups.

Gross findings in dying and sacrificed rats of the two top doses showed congested and speckled livers and hydronephrosis, hydroureter, ureteral obstructions, hematuria in the urinary tract. Bilateral atrophy of the testes was found in males of all dose groups. Microscopically, sinusoidal congestion and fatty degeneration of centrilobular hepatocytes were found (the ‘fatty’ nature was not confirmed by specific staining procedures). Hydronephrosis was confirmed by histopathological examination for males at ≥3160 ppm and female rats at 10000 ppm. Intra-luminal cell debris, necrosis of the tubular epithelium, and papillary necrosis were reported as the causes of the obstructive urinary tract lesions.

Renal tubular necrosis and papillary necrosis was evident in treated male and female rats of all dose groups. The testes atrophy was related to degenerated epithelium of seminiferous tubules.

A NOAEL could not be determined in this early study; 100 ppm (6 mg/kg bw/d for male rat, 8 mg/kg bw/d for female rats) is the LOAEL for oral subchronic administration of 4-tertbutylbenzoic acid.

Conclusions:
According to the results in this study the oral LOAEL of the test substance is 100 ppm (6 mg/kg in male rats, 8 mg/kg in female rats).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable reference

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: data is contained in EU risk assessment report of the substance - credibility is assumed. Reference to origin (HRC (1994, 1995))
Reference:
Composition 0
Composition 0
Composition 0
Principles of method if other than guideline:
5-day and 28-d repeated dose inhalation toxicity study using rats (male/female), snout-only exposition to test substance, subsequent examination of substance-induced effects
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
inhalation
Type of inhalation exposure:
other: snout-only
Vehicle:
air
Critical effects observed:
not specified

There were no clinical signs, bodyweight changes, effects on food and water consumption, macroscopic pathology findings or differences in organ weights that were considered to be attributable to exposure to PTBBA. Samples of a number of organs were preserved, but not prepared for microscopic pathology (HRC, 1994).

Liver weights of high dose females were significantly higher than control values (+9%). There were no other clinical signs, bodyweight changes, effects on food consumption, macroscopic or microscopic changes in main study rats that were attributable to p-tert butyl benzoic acid. Behavioural observations revealed a slight increase in the incidence of body tremor in the low and high dose group males after 1 week of exposure. After 4 weeks the incidence of body tremor was increased for high dose males. Among high dose males, there was a significant decrease in activity counts with tendency towards decreased rearing counts. Also, facial staining and hair loss occurred with slightly increased frequency in high dose males. The number of males with decreased arousal and urinating/defecating while in the arena was increased in the mid and high dose groups. No similar findings were noted among treated females. Neither the microscopic examination of the organs examined in the main study nor the examination of the nervous tissues in satellite rats revealed any lesions, which were attributable to 4-tert-butylbenzoic acid. The occurrence of body tremor might be considered as the most sensitive and earliest neurobehavioural effect. Since no behavioural change was noted for low dose males after 4 weeks of exposure and no body tremor was observed for mid dose males, the NOAEC was considered to be 5 mg/m³ for male rats. The authors proposed a NOAEC of 15 mg/m³ for female rats. Based on the knowledge that the liver was a target organ in other repeated dose studies, the rapporteur’s opinion is that due to increased liver weight 5 mg/m³ should also be considered as the NOAEC for female rats. (HRC, 1995)

Conclusions:
According to the results of this study the inhalation NOAEC for the test substance is 5 mg/m³.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable reference

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: data contained in EU risk assessment report of the substance - credibility is assumed. Reference to origin (Shell Research Ltd. London)
Reference:
Composition 0
Composition 0
Principles of method if other than guideline:
28 d repeated dose dermal toxicity study in rats using different concentrations of the test substance in DMSO
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rat
Strain:
other: Carworth Farm E
Sex:
male/female
Type of coverage:
other: animals received solutions of the substance topically on shaved skin
Vehicle:
DMSO
Duration of treatment / exposure:
28 d
Remarks:
Doses / Concentrations:
0, 7.5, 15, 30 and 60 mg/kg bw/d
Basis:
no data
No. of animals per sex per dose:
8 males; 8 females
Critical effects observed:
not specified

Growth rates were reduced in males and during the first two weeks in female rats exposed to 30 and 60 mg/kg bw/d resulting in significantly lower final body weight of males of these dose groups. Dose-related significant increases in absolute and relative liver weights were seen in female rats of all dose groups (+11, 23, 27, 30%) and in male rats exposed to 15 mg/kg/d and above (+8, 11, 17%). Increased relative weights of kidneys were observed in two top doses of female rats, and decrease in relative and absolute testes weights were determined for male rats receiving 60 mg/kg/d. Histopathology of the testes revealed a degeneration of germinal epithelium in males exposed to 60 mg/kg/d. No other toxic effect was observed in the liver and the kidneys of the four animals/sex/group examined except an increased basophilia of centrilobular hepatocytes that was considered of uncertain significance. The LOAEL was 7.5 mg/kg bw/d. This study was flawed by the small numbers of test parameters and animals and a poor documentation (summary, 2 tables and 1 figure available).

Conclusions:
According to the results of this study the dermal LOAEL for the test substance is 7.5 mg/kg.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
7.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable reference

Mode of Action Analysis / Human Relevance Framework

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
90 day oral study with large set of analysis (urinalysis, hematology, clinical chemistry, gross and microscopic examinations were performed)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The determined NOAEC was the basis for the DNEL (inhalation) derivation.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The determined LOAEL was the basis for the DNEL (dermal) derivation.

Justification for classification or non-classification

Comments in the EU risk assessment report (p. 63-65):

Oral 90 d study: 6 mg/kg PTBBA (= LOAEL) producing kidney and testis toxicity is markedly lower than the critical concentration of 50 mg/kg bw/d for harmful/R48/22.

Dermal 90 d study:

Although rats of this study were dermal exposed to preparations of 4-tert-butylbenzoic acid and DEA, a contribution of DEA, especially on the liver metabolism can not be ruled out. In view of the observation, that effects in the same target organs were similar to those mentioned after repeated oral or inhalation exposures they were attributed to 4-tert-butylbenzoic acid.

Dermal 28 day study:

This study was flawed by the small numbers of test parameters and animals and a poor documentation (summary, 2 tables and 1 figure available). LOAEL =7.5 mg/kd bw/d corresponds to 2.3 mg/kg bw/d in a 90 day study, which is markedly lower than the critical dose of 100 mg/kg bw/d for harmful/R48/21. This is supported by the LOAEC of the 7/13 week study of Cagen et al., 1989, where LOAEC was 17.5 mg/kg bw/d (the lowest dose tested) was significantly below the limit concentration of 100 mg/kg bw/d.

Inhalation 10/11 day study:

The main weaknesses of this study were its short exposure duration, the lack of data from neurofunctional testing battery, lack of water and feed consumption data and urinalysis, the restricted panel of organs processed for histopathology. However, data wasdeemed to give valid information on the toxicity of the test substance.Using Haber’s rule to extrapolate to a 90 day study design, LOAEC = 12.5 mg/m³ of PTBBA corresponds to 1.5 mg/ m³ which is 166-fold below the limit concentration for harmful/R48/20.

Based on the above data, 4-tert-butylbenzoic acid should be classified “T, R48/23/24/25,

Toxic by inhalation, in contact with skin and if swallowed”. The adverse effect levels were

far below the guidance values for the classification as harmful. Therefore the currently

applied classification should be replaced. In September 2007 the TC C&L agreed T;

R48/23/24/25.

Discussion on target organ toxicity:

Growth retardation:

The observation that feed consumption was not changed by treatment (Cagen et al., 1989) or reduction in feed consumption was seen only in high doses of 4-tertbutylbenzoic acid (Hunter et al., 1965) support the conclusion that reduced gain of body weight and reduction of final body weight can be interpreted indicative for non-specific toxic effect of PTBBA.

Neurotoxicity:

Regional poliomyelomalacia and responsive gliosis of the spinal cord described in the Shell study (1982) can be associated to the fore and hind limb paralysis and gait abnormalities that was observed in the 11 day-inhalation study at particle concentrations of 106 mg/m³ and above. Similar lesions might be expected for animals with hind limb paralysis receiving diet concentrations of 1000 ppm and above of the 90 day study (Hunter et al., 1965). The fact that nervous tissue damage has not been observed in the dermal study is no proof for the absence of neurological effects since methods applied in all repeat-dose studies are routine staining procedures which may be insufficient to detect specific lesions in cellular compartments of the nervous system.

Urinary tract toxicity:

4-tert-Butylbenzoic acid affected the urinary system by all exposure routes. The tubular epithelium of the distal cortical convoluted tubules and papillary region (renal pelvis) seemed to be the primary sites of 4-tert-butylbenzoic acid toxicity. Increased diuresis, hematuria, tubular casts, regenerative epithelium, interstitial inflammation, hyrdronephrosis and hydroureter were associated lesions that can be considered as the death-related cause in the oral study of Hunter et al. (1965).

Liver toxicity:

Increased activity of serum transaminases (Shell, 1982), speckled, enlarged appearance of the liver were consistent with the liver cell toxicity observed in all repeat-dose studies available. The increase in liver weights were considered as indicative for hepatotoxicity in those studies (HRC, 1995, Shell, 1975), where overt morphological lesions or biochemical findings could not be observed or were unknown due to the lack of examination since hepatocyte cytotoxicity in other studies were associated to increased liver weights in the other studies. Reduced serum cholesterol levels and fatty vacuolation of liver cells can be assumed to reflect a disturbance of lipid metabolism. This assumption was supported by in vitro data on isolated hepatocytes showing that PTBBA inhibited fatty acid synthesis and increased medium and long chain acyl CoA esters (McCune et al., 1982).

Toxicity in reproductive organs:

Testicular lesions attributable to 4-tert-butylbenzoic acid occurred in rats exposed via all exposure routes. Similar effects were observed in the studies available, which were characterised by the degeneration of germinal epithelium resulting in disturbance of spermatogenesis at several stages of spermatogenic cells. The presence of multinucleated giant cells in the luminal of seminiferous tubules of testes was indicative for a more chronic process. Corresponding secondary changes were atrophy and inflammatory responses of the epididymides.

Toxic effects on the hemopoietic system:

Signs of microcytic hypochromic anemia were found at diet concentration of 10000 ppm (Hunter et al., 1965), at particle concentration of 525 mg/m³ (Shell, 1982) and at 70 mg/kg bw/d of 4-tert-butylbenzoic acid applied topically (Cagen et al., 1989). Indications for increased erythrocyte destruction were not identified, other causes might be more likely, however unknown.

Increased WBC counts and increased percentages of neutrophilic granulocytes observed at 525 mg/m³ of the inhalation study of Shell (1982) and at 10000 ppm of the oral study of Hunter and his colleagues (1965) were presumably related to inflammatory responses to the damage in target organs.

Immunotoxicity:

Due to the scarce database the toxicological significance of cortical atrophy of the thymus following lymphocytolysis remains uncertain (Shell, 1982). Most of the rats affected were those dying spontaneously.

Classification according to EC No. 1272/2008 (Committee for Risk Assessment RAC, ECHA/RAC/CLH-O-0000001579-64-01/A1, Adopted 21 February 2011):

STOT Rep 1 - H372